DNA sequences encoding polypeptides having β-1,3-glucanase activity

ABSTRACT

The present invention provides chemically regulatable DNA sequences capable of regulating transcription of an associated DNA sequence in plants or plant tissues, chimeric constructions containing such sequences, vectors containing such sequences and chimeric constructions, and transgenic plants and plant tissues containing these chimeric constructions. In one aspect, the chemically regulatable DNA sequences of the invention are derived from the 5′ region of genes encoding pathogenisis-related (PR) proteins. The present invention also provides anti-pathogenic sequences derived from novel cDNAs coding for PR proteins which can be genetically engineered and transformed into plants to confer enhanced resistance to disease. Also provided is a method for the exogenous regulation of gene expression in plants, which comprises obtaining a plant incapable of regulating at least one gene or gene family, or at least one heterologous gene, due to the deactivation of at least one endogenous signal transduction cascade which regulates the gene in the plant, and applying a chemical regulator to the plant at a time when expression of the gene is desired. A novel signal peptide sequence and corresponding DNA coding sequence is also provided. Further provided are assays for the identification and isolation of additional chemically regulatable DNA sequences and cDNAs encoding PR proteins and assays for identifying chemicals capable of exogenously regulating the chemically regulatable DNA sequences of the invention.

CROSS-REFERENCE TO RELATED U.S. PATENT APPLICATIONS

This application is a continuation of application Ser. No. 08/971,217,filed Nov. 14, 1997, issued as U.S. Pat. No. 5,942,662, which is acontinuation of application Ser. No. 08/457,364, filed May 31, 1995,issued as U.S. Pat. No. 5,847,258, which is a divisional of applicationSer. No. 08/181,271, filed Jan. 13, 1994, issued as U.S. Pat. No.5,614,395, which is a continuation-in-part of abandoned application Ser.No. 08/093,301, filed Jul. 16, 1993, which is a continuation ofabandoned application Ser. No. 07/973,197, filed Nov. 6, 1992, which isa continuation of abandoned application Ser. No. 07/678,378, filed Apr.1, 1991, which is a continuation of abandoned application Ser. No.07/305,566, filed Feb. 6, 1989, which is a continuation-in-part ofabandoned application Ser. No. 07/165,667, filed Mar. 8, 1988. Saidapplication Ser. No. 08/181,271 filed Jan 13, 1994 (U.S. Pat. No.5,614,395) is also a continuation-in-part of abandoned application Ser.No. 08/042,847, filed Apr. 6, 1993, which is a continuation of abandonedapplication Ser. No. 07/632,441, filed Dec. 21, 1990 which is acontinuation-in-part of abandoned application Ser. Nos. 07/425,504 and07/165,667, filed Oct. 20, 1989 and Mar. 8, 1988, respectively. Saidapplication Ser. No. 08/181,271 filed Jan. 13, 1994 (U.S. Pat. No.5,614,395) is also a continuation-in-part of abandoned application Ser.No. 07/848,506, filed Mar. 6, 1992, which is a continuation-in-part ofabandoned application Ser. No. 07/768,122, filed Sep. 27, 1991, which isa continuation-in-part of abandoned application Ser. No. 07/580,431,filed Sep. 7, 1990, which is a continuation-in-part of abandonedapplication Ser. No. 07/425,504, filed Oct. 20, 1989, which is acontinuation-in-part of abandoned application Ser. No.07/368,672, filedJun. 20, 1989, which is a continuation-in-part of abandoned applicationSer. No. 07/329,018, filed Mar. 24, 1989. Said abandoned applicationSer. No. 07/425,504, filed Oct. 20, 1989, is also a continuation-in-partof abandoned application Ser. No. 07/381,443, filed Jul. 18, 1989, whichis a continuation-in-part of abandoned application Ser. No. 07/353,312,filed May 17, 1989, which is a continuation-in-part of abandonedapplication Ser. No. 07/226,303, filed Jul. 29, 1988. Said applicationSer. No. 08/181,271 filed Jan. 13, 1994 (U.S. Pat. No. 5,614,395) isalso a continuation-in-part of abandoned application Ser. No.08/045,957, filed Apr. 12, 1993.

FIELD OF THE INVENTION

One aspect of the present invention relates to the chemical regulationof gene expression. In particular, this aspect relates to non-coding DNAsequences which, in the presence of chemical regulators, regulate thetranscription of associated DNA sequences in plants. Another aspect ofthe invention relates to DNA molecules encoding proteins capable ofconferring plant disease and/or plant pest resistance. Both aspects ofthe invention relate, in part, to genes associated with the response ofplants to pathogens.

BACKGROUND OF THE INVENTION

Advances in recombinant DNA technology coupled with advances in planttransformation and regeneration technology have made it possible tointroduce new genetic material into plant cells, plants or plant tissue,thus introducing new traits, e.g., phenotypes, that enhance the value ofthe plant or plant tissue. Recent demonstrations of geneticallyengineered plants resistant to pathogens (EP-A 240 332 and EP-A 223 452)or insects (Vaeck, M. et al., Nature 328: 33 (1987)) and the productionof herbicide tolerant plants (DeBlock, M. et al., EMBO J. 6: 2513(1987)) highlight the potential for crop improvement. The target cropscan range from trees and shrubs to ornamental flowers and field crops.Indeed, it is clear that the “crop” can also be a culture of planttissue grown in a bioreactor as a source for some natural product.

A. General Overview of Plant Transformation Technology

Various methods are known in the art to accomplish the genetictransformation of plants and plant tissues (i.e., the stableintroduction of foreign DNA into plants). These include transformationby Agrobacterium species and transformation by direct gene transfer.

1. Agrobacterium-Medicated Transformations

A. tumefaciens is the etiologic agent of crown gall, a disease of a widerange of dicotyledons and gymnosperms, that results in the formation oftumors or galls in plant tissue at the site of infection. Agrobacterium,which normally infects the plant at wound sites, carries a largeextrachromosomal element called the Ti (tumor-inducing) plasmid.

Ti plasmids contain two regions required for tumorigenicity. One regionis the T-DNA (transferred-DNA) which is the DNA sequence that isultimately found stably transferred to plant genomic DNA. The otherregion rquired for tumorigenicity is the vir (virulence) region whichhas been implicated in the transfer mechanism. Although the vir regionis absolutely required for stable transformation, the vir DNA is notactually transferred to the infected plant. Transformation of plantcells mediated by infection with Agrobacterium tumefaciens andsubsequent transfer of the T-DNA alone have been well documented. See,for example, Bevan, M. W. and Chilton, M-D., Int. Rev. Genet. 16: 357(1982).

After several years of intense research in many laboratories, theAgobacterium system has been developed to permit routine transformationof a variety of plant tissue. Representative species trnmsformed in thismanner include tobacco, tomato, sunflower, cotton, rapeseed, potato,soybean, and poplar. While the host range for Ti plasmid transformatonusing A. tumefaciens as the infecting agent is known to be very large,tobacco has been a host of choice in laboratory experiments because ofits ease of manipulation.

Agrobacterium rhizogenes has also been used as a vector for planttransformation. This bacterium, which incites hairy root formation inmany dicotyledonous plant species, carries a large extrachromosomalelement called an Ri (root-inducing) plasmid which functions in a manneranalogous to the Ti plasmid of A. tumefaciens. Transformation using A.rhizogenes has developed analogously to that of A. tumefaciens and hasbeen successfully utilized to transform, for example, alfalfa, Solanumnigrum L., and poplar.

2. Direct Gene Transfer

Several so-called direct gene transfer procedures have been developed totransform plants and plant tissues without the use of an Agrobacteriumintermediate (see, for example, Koziel et al., Biotechnology 11: 194-200(1993); U.S. application Ser. No. 08/008,374, filed Jan. 25, 1993,herein incorporated by reference in its entirety). In the directtransformation of protoplasts the uptake of exogenous genetic materialinto a protoplast may be enhanced by use of a chemical agent or electricfield. The exogenous material may then be integrated into the nucleargenome. The early work was conducted in the dicot tobacco where it wasshown that the foreign DNA was incorporated and transmitted to progenyplants, see e.g. Paszkowski, J. et al., EMBO J. 3: 2717 (1984); andPotrykus, I. et al., Mol. Gen. Genet. 199: 169 (1985).

Monocot protoplasts have also been transformed by this procedure in, forexample, Triticum monococcum, Lolium multiflorum (Italian ryegrass),maize, and Black Mexican sweet corn.

Alternatively exogenous DNA can be introduced into cells or protoplastsby microinjection. A solution of plasmid DNA is injected directy intothe cell with a finely pulled glass needle. In this manner alfalfaprotoplasts have been transformed by a variety of plasmids, see e.g.Reich, T. J. et al., Bio/Technology 4: 1001 (1986).

A more recently developed procedure for direct gene transfer involvesbombardment of cells by microprojectiles carrying DNA, see Klein, T. M.et al., Nature 327: 70 (1987). In this procedure tungsten particlescoated with the exogenous DNA are accelerated toward the target cells,resulting in at least transient expression in the example reported(onion).

B. Regeneration of Transformed Plant Tissue

Just as there is a variety of methods for the transformation of planttissue, there is a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated. In recent years it has become possible to regenerate manyspecies of plants from callus tissue derived from plant explants. Theplants which can be regenerated from callus include monocots, such ascorn, rice, barley, wheat and rye, and dicots, such as sunflower,soybean, cotton, rapeseed and tobacco.

Regeneration of plants from tissue transformed with A. tumefaciens hasbeen demonstrated for several species of plants. These includesunflower, tomato, white clover, rapeseed, cotton, tobacco, and poplar.The regeneration of alfalfa from tissue transformed with A. rhizogeneshas also been demonstrated. Plant regeneration from protoplasts is aparticularly useful technique, see Evans, D. A. et al., in: “Handbook ofPlant Cell Culture”, Vol. 1, MacMillan Publ. Co., 1983, p. 124. When aplant species can be regenerated from protoplasts, then direct genetransfer procedures can be utilized, and transformation is not dependenton the use of A. tumefaciens. Regeneration of plants from protoplastshas been demonstrated for rice, tobacco, rapeseed, potato, eggplant,cucumber, poplar, and corn.

Various plant tissues may be utilized for transformation with foreignDNA. For instance, cotyledon shoot cultures of tomato have been utilizedfor Agrobacterium mediated transformation and regeneration of plants(see European application 0249432). Further examples include Brassicaspecies (see WO 87/07299) and woody plant species, particularly poplar(see U.S. Pat. No. 4,795,855, incorporated by reference herein in itsentirety).

The technological advances in plant transformation and regenerationtechnology highlight the potential for crop improvement via geneticengineering. There have been reports of genetically engineered tobaccoand tomato plants which are resistant to infections of, for example,tobacco mosaic virus (TMV) and resistant to different classes ofherbicides. Insect resistance has been engineered in tobacco and tomatoplants.

C. Cell Cultures

The potential for genetic engineering is not limited to field crops butincludes improvements in ornamentals, forage crops and trees. A lessobvious goal for plant biotechnology, which includes both geneticengineering and tissue culture applications, is the enhanced productionof a vast array of plant-derived chemical compounds including flavors,fragrances, pigments, natural sweeteners, industrial feedstocks,antimicrobials and pharmacuticals. In most instances these compoundsbelong to a rather broad metabolic group, collectively denoted assecondary products. Plants may produce such secondary products to wardoff potential predators, attract pollinators, or combat infectiousdiseases.

Plant cell cultures can be established from an impressive array of plantspecies and may be propagated in a bioreactor. Typical plant speciesinclude most of those that produce secondary products of commercialinterest. It has been clearly demonstrated in a number of agriculturallyimportant crop plants that stable genetic variants arising from thetissue culture of plant somatic cells (somaclonal variation) can beinduced and selected. Numerous advantages flow from plant tissue cultureproduction of secondary compounds. These include (1) the possibility ofincreased purity of the resultant product, (2) the conversion ofinexpensive precursors into expensive end products by biotransformation,and (3) the potential for feeding substrate analogs to the culture tocreate novel compounds.

D. Advantages of Controlled Gene Expression

Whether the target of genetic engineering of plants is a field crop,ornamental shrub, flower, tree or a tissue culture for use in abioreactor, a principal advantage to be realized is the control ofexpression of the chimeric gene so that it is expressed only at theappropriate time and to the appropriate extent, and in some situationsin particular parts of the plant. For example, in order to achieve adesirable phenotype the chimeric gene may need to be expressed at levelsof 1% of the total protein or higher. This may well be the case forfungal resistance due to chimeric chitinase expression or insectresistance due to increased proteinase inhibitor expression. In thesecases the energy expended to produce high levels of the foreign proteinmay result in a detriment to the plant whereas, if the gene wereexpressed only when desired, for instance when a fungal or insectinfestation is imminent, the drain on energy, and therefore yield, couldbe reduced.

Alternatively, the phenotype expressed by the chimeric gene could resultin adverse effects to the plant if expressed at inappropriate timesduring development. For example, if the chimeric gene product were aplant hormone that induced pod abscission, early expression could bringabout abscission of the fruit from the plant before the seed hadmatured, resulting in decreased yield. In this case it would be muchmore advantageous to induce the expression of this type of gene to atime when pod abscission is preferred, or least injurious to the plant.

For tissue in culture or in a bioreactor the untimely production of asecondary product could lead to a decrease in the growth rate of theculture resulting in a decrease in the yield of the product. Therefore,it would be advantageous to allow the culture to grow without expressingthe secondary product and then induce the chimeric gene at anappropriate time to allow for an optimized expression of the desiredproduct.

In view of considerations like these, as well as others, it is clearthat control of the time, extent and/or site of expression of thechimeric gene in plants or plant tissues would be highly desirable.Control that could be exercised easily in a field, a greenhouse or abioreactor would be of particular commercial value.

E. Known Regulatable Gene Expression Systems in Plants

Several plant genes are known to be induced by various internal andexternal factors including plant hormones, heat shock, chemicals,pathogens, lack of oxygen and light. While few of these systems havebeen described in detail, in the best characterized, an increasedaccumulation of mRNA leads to an increased level of specific proteinproduct.

As an example of gene regulation by a plant hormone, abscissic acid(ABA) has been shown to induce the late embryogenesis abundant mRNAs ofcotton, see Galau, G. A. et al., Plant Mol. Biol. 7: 155 (1986). Inanother example, gibberellic acid (GA3) induces malate synthasetranscripts in castor bean seeds and alpha-amylase isozymes in barleyaleurone layers, see Rodriguez, D. et al., Plant Mol. Biol. 9: 227(1987); Nolan, R. C. et al., Plant Mol. Biol. 8: 13 (1987).

The regulation of heat shock protein genes of soybean has been studiedin detail. Treatment of plants for several hours at 40° C. results inthe de novo synthesis of several so-called heat shock proteins (Key, J.et al., Proc. Natl. Acad. Sci. USA, 78: 3526 (1981)). Several such geneshave been isolated and their regulation studied in detail. Theexpression of these genes is primarily controlled at the level oftranscription. The promoter of the hsp70 gene has been fused to theneomycin phosphotransferase II (NptII) gene and the chimeric gene hasbeen shown to be induced by heat shock (Spena, A. et al., EMBO J. 4:2736 (1985)) albeit at a lower level than the endogenous heat shockgenes.

Another class of inducible genes in plants include the light regulatedgenes, most notably the nuclear encoded gene for the small subunit ofribulose 1,5-bisphosphate carboxylase (RUBISCO). Morelli, G. et al.,Nature 315: 200 (1985)) and Hererra-Estrella, L. et al., Nature 310: 115(1984)) have demonstrated that the 5′ flanking sequences of a peaRUBISCO gene can confer light inducibility to a reporter gene whenattached in a chimeric fashion. This observation has been extended toother light inducible genes such as the chlorophyll a/b binding protein.

The alcohol dehydrogenase (adh) genes of maize have been extensivelystudied. The adh1-s gene from maize was isolated and a portion of the 5′flanking DNA was shown to be capable of inducing the expression of achimeric reporter gene (e.g., chloramphenicol acetyl transferase, CAT)when the transiently transformed tissue was subjected to anaerobicconditions (Howard, E. et al., Planta 170: 535 (1987)).

A group of chemicals known as safeners have been developed to protect or“safen” crops against potentially injurious applications of herbicides.While a general mechanism for the action of such compounds has not beenfully developed, regulation of naturally regulatable genes by suchcompounds is one possible mechanism. It has recently been reported thathigher levels of a glutathione-S-transferase (GST) are induced in maizetreated with the safener2-chloro-4-(trifluoromethyl)-5-methyl-thiazolecarboxylic acid benzylester, see Wiegand, R. C. et al., Plant Mol. Biol. 7: 235 (1986).Although the level of GST mRNA is elevated upon treatment with thesafener, the mechanism leading to the elevation was not reported.

Many plants, when reacting hypersensitively toward various pathogens,are stimulated to produce a group of acid-extractable, low molecularweight pathogenesis-related (PR) proteins (Van Loon, L. C., Plant Mol.Biol. 4: 111 (1985)). Of particular interest, however, is theobservation that these same PR proteins accumulate to high levels inplants treated with chemicals such as polyacrylic acid andacetylsalicylic acid (Gianinazzi, S. et al., J. Gen. Virol. 23: 1(1974); White, R. F., Virology 99: 410 (1979)). The presence of PRproteins has been correlated with the induction of both a local andsystemic resistance against a broad range of pathogens. An interspecifictobacco hybrid resistant to tobacco mosaic virus (TMV) was shown toexpress the PR-proteins constitutively (Ah1, P. et al., Plant Sci. Lett.26: 173 (1982)). Furthermore, immunoprecipitation of in vitrotranslation products using mRNA from either TMV-infected or chemicallytreated tobacco (Cornelissen, B. J. C. et al., EMBO J. 5: 37 (1986);Carr, J. P. et al., Proc. Natl. Acad. Sci. USA 82: 7999 (1985))indicated that the increased level of PR-protein was a result of RNAaccumulation. Therefore, induction of PR protein genes by chemicals orpathogens provides a method to address the problem of chemicallyregulating gene expression in plants and plant tissue.

F. Chemical Regulation of Expression

In some cases it will be desirable to control the time and/or extent ofthe expression of introduced genetic material in plants, plant cells orplant tissue. An ideal situation would be the regulation of expressionof an engineered trait at will via a regulating intermediate that couldbe easily applied to field crops, ornamental shrubs, bioreactors, etc.This situation can now be realized by the present invention which isdirected to, among other things, a chemically regulatable chimric geneexpression system comprising a chemically regulatable, non-coding DNAsequence coupled, for example, to a gene encoding a phenotypic trait,such that the expression of that trait is under the control of theregulator, e.g. such that expression from the regulated gene isdetermined by the presence or absence of a chemical regulator. Thissystem is the first demonstration of the concept of chemical regulationof chimeric gene expression in plants or plant tissue. As such itenables the production of transgenic plants or plant tissue and thecontrol of traits expressed as a function of a chemical regulator.

The present invention also teaches the external manipulation of theexpression of endogenous genes which contain chemically regulatablesequences by the application of a chemical regulator (see Ward, E. etal., Plant Cell 3: 1085-1094 (1991); Williams et al., Bio/Technology 10:540-543 (1992); and Uknes, S. et al., Plant Cell 5: 159-169 (1993). Thecontrol provided is somewhat limited, however, due to the responsivenessof such sequences to endogenous chemical metabolites and cell signals aswell as externally applied chemical regulators. In yet another aspect ofthe invention, alterations are taught which block the responsiveness ofthese genes to endogenous signals while maintaining responsiveness toexternally applied chemical regulators.

G. Insect Resistance

Pest infestation of crop plants causes considerable loss of yieldthroughout the world and most crops grown in the U.S. sufferinfestation, particularly from insects. Major insect pests in the U.S.include the European Corn Borer (Ostrinia nubilalis) in maize, thecotton bollworm Heliothis zea) and the pink bollworm (Pectinophoragossypiella) in cotton and the tobacco budworm (Heliothis virescens) intobacco. Resistance to pests is difficult to achieve using conventionalbreeding programs and typically pests have been controlled usingchemical pesticides.

Recent advances in molecular biology and plant transformation technologyhave demonstrated the possibility of expressing in transgenic plantsgenes encoding insecticidal proteins; this represents a novel approachin the production of crop plants resistant to pests. Most notably, theexpression of genes encoding the Bacillus thuringiensis δ-endotoxin hasbeen successful in a wide range of plant species, and the analysis oftransgenic lines expressing such genes has been well documented (Vaecket al., Nature 328: 33-37 (1987); Fischoff et al., Biotechnology 5:807-813 (1987); Carozzi et al., Plant Mol. Biol. 20: 539-548 (1992);Koziel et al., Biotechnology 11: 194-200 (1993)). Other insecticidalgenes have been used successfully in generating insect resistanttransgenic plants.

One approach has been the overexpression of genes encoding insect enzymeinhibitors such as trypsin inhibitors or seed proteins with knowninsecticidal activity, such as lectins (Hilder et al., Nature 330:160-163 (1987)). Indeed, plants expressing both the cowpea trypsininhibitor and pea lectin were shown to have additive effects inproviding insect resistance (Boulter et al., Crop Protection 9: 351-354(1990)). In cases where pests are able infest parts of the plant ortissues not readily accessible to conventional pesticides, a transgenicapproach may be more successfiul than the use of conventionalpesticides.

For example, the tobacco budworm Heliothis is well known to be difficultto control using pesticides because it burrows deep into the planttissue. Additionally, some pests of roots, such as nematodes, are notreadily controlled by foliar applications of pesticides. An advantage inthe use of taansgenic plants expressing insecticidal proteins is thecontrolled expression of the proteins in all desired tissues.

Chitinases catalyze the hydrolysis of chitin, a β-1,4-linked homopolymerof N-acetyl-D-glucosamine. Several different plant chitinases have beendescribed and the cDNA sequences for some of these have been reported(Meins et al., Mol. Gen. Genet. 232: 460-469 (1992)). Based onstructural characteristics, three classes have been distinguished. ClassI chitinases have two structural domains, a cysteine-rich amino-terminalhevein domain and a carboxyterminal catalytic domain separated from theformer by a variable spacer. Class II chitinases lack the cysteine-richhevein domain and all or part of the variable spacer, but retain thecatalytic domain. Class III chitinases lack the hevein domain andcontain a catalytic domain that shares no significant homology with thatof the class I or class II enzymes.

Class I chitinase gene expression is induced by ethylene, whereas classII and class III chitinase gene expression is induced in the SARresponse. The chitinase/lysozyme disclosed in U.S. application Ser. No.07/329,018 and the chitinase/lysozymes disclosed in U.S. applicationSer. No. 07/580,431 (provided herein as SEQ ID Nos. 29 and 30,respectively) are class III chitinases. It is well known that the levelof chitinase activity of plants increases dramatically after pathogeninvasion (Mauch et al., Plant Physiol. 76: 607-611 (1984)) and this ispresumably due to the host plant's attempts to degrade the chitin of thefungal cell wall. Furthermore, chitinase has been shown in vitro toinhibit fungal and insect growth, and in transgenic plants a bacterialchitinase has been shown to exhibit inhibitory effects towards numerouspathogens and pests including insects (Suslow & Jones WPO 90-231246;U.S. Pat. Nos. 4,940,840 and 4,751,081; herein incorporated by referencein their entirety).

H. Resistance Response of Plants to Pathogen Infection

For over 90 years, scientists and naturalists have observed that whenplants survive pathogen infection they develop an increased resistanceto subsequent infections. In 1933, a phenomenon termed “physiologicalacquired immunity” was descibed in an extensive literature review byChester, K. S., Quart. Rev. Biol. 8: 275-324 (1933). At that time,scientists believed they were investigating a phenomenon analogous tothe immune response in mammals. In retrospect, at least teree differentprocesses were being called “aquired immunity”: viral cross protection,antagonism (or biocontrol), and what we now refer to as systemicacquired resistance (SAR).

1. Systemic Acquired Resistance (SAR)

The first systematic study of SAR was published by A. Frank Ross in1961. Using tobacco mosaic virus (TMV) on local lesion hosts, Rossdemonstrated that infections of TMV were restricted by a priorinfection. This resistance was effective against not only TMV, but alsotobacco necrosis virus and certain bacterial pathogens. Ross coined theterm “systemic acquired resistance” to refer to the inducible systemicresistance (Ross, A. F., Virology 14: 340-358 (1961)) and “localizedacquired resistance” (LAR) to describe the resistance induced ininoculated leaves (Ross, A. F., Virology 14: 329-339 (1961)). It isstill unclear whether SAR and LAR are aspects of the same response ordistinct processes.

In the past 30 years, SAR has been demonstrated in many plant speciesand the spectrum of resistance has been broadened to include not onlyviruses and bacteria, but also many agronomically importantphytopathogenic fungi (see Kuc, J., BioScience 32: 854-860 (1982).However, understanding of the biochemical events leading to theestablishment of SAR had not progressed substantially until the pastdozen years. In 1982, the accumulation of a group of extracellularproteins called pathogenesis-related (PR) proteins were reported tocorrelate with the onset of SAR (Van Loon, L. C. et al., Neth. J. Plant.Path. 88: 237-256 (1982)). In 1979, salicylic acid (SA) and certainbenzoic acid derivatives were reported to be able to induce bothresistance and the accumulation of PR proteins (White, R. F., Virology99: 410-412 (1979). As a result, SA was considered as a possibleendogenous signal molecule (Van Loon, L. C. et al., Neth. J. Plant.Path. 88: 237-256 (1982)). However, progress slowed through the 1980'sand the involvement of PR proteins and salicylic acid in SAR wasquestioned.

With the advent of genetic engineering and recombinant DNA technology,the possibility of manipulating genetic material to improve thephenotype of plants has arisen. The present invention is based in partupon the discovery of the identity and role of genes involved in SARwhich has allowed the application of modern molecular biologicaltechniques for improved plant disease and plant pest resistance.

SUMMARY OF THE INVENTION

There are two major aspects of the present invention. The first aspectrelates to chemically regulatable DNA sequences and the chemicals whichregulate them. The second aspect relates to plant pathogenesis-relatedproteins. Both aspects of the invention have arisen, in part, from theinventors' identification and characterization of cDNAs andcorresponding genes involved in the plant response to pathogeninfection.

A principal object of the present invention is to provide a means forchemically regulating the expression of a desired gene in a plant, seed,or plant tissue.

To meet this objective, the first aspect of the present inventionincludes: (a) chemically regulatable DNA sequences, preferably insubstantially pure form; (b) one or more chemically regulatable DNAsequences in combination with one or more parts but not all of anycoding DNA sequences with which the regulatable sequences are associatedin naturally occurring genes; (c) chimeric genes containing one or morechemically regulatable DNA sequences; (d) vectors containing sequencesor genes of (a), (b) and/or (c); (e) plants, seeds, and plant tissuecontaining the chemically regulatable chimeric genes; and (f) chemicalregulation of chemically regulatable chimeric genes in plant tissue. Theinvention further includes a signal peptide, a DNA sequence coding forthe signal peptide, and the substantially pure forms of severalnaturally occurring chemically inducible genes.

The first aspect of the invention further embraces several uses of thechemically regulatable DNA sequences: (a) regulation of chimeric genesin cells propagated in a bioreactor, (b) an assay for chemicalregulators, (c) developmental regulation of the plant, (d) regulation ofplant sterility and (e) regulation of chimeric and/or heterologous geneexpression in a transformed plant. Other uses and advantages will beapparent from the following detailed description of the invention.

Another principal object of the present invention is to providetransgenic plants expressing levels of plant pathogenesis-relatedproteins, or substantially homologous proteins, which confer an enhanceddisease-resistant and/or pest-resistant phenotype with respect towild-type plants.

Accordingly, to meet this objective and others, the second aspect of thepresent invention disclosed herein provides for the isolation, cloningand identification of novel cDNA clones coding for plantpathogenesis-related (PR) proteins. These cDNAs, or their genomiccounterparts, or DNA molecules with substantial homology to either (allof the above collectively referred to herein as “anti-pathogenicsequences”), can be engineered for expression of the encoded PR proteinsor anti-sense RNA and transformed into plants to confer enhancedresistance or tolerance to various diseases and/or pests as describedherein. These DNA molecules may be engineered for constitutiveexpression, expression in particular tissues or at certain developmentalstages, or induced expression in response to an inducer, for example inresponse to a chemical inducer as described herein.

The present invention further provides novel methods for differentialscreening and enriching for induced cDNAs, particularly those cDNAsinduced in response to pathogen infection or a chemical inducer whichtriggers a response mimicking pathogen infection.

The present invention is further drawn to a method of exogenouslycontrolling the regulation of gene expression in plants. The methodinvolves altering a plant to inactivate a predetermined signaltransduction cascade, and subsequently treating the thus-modified plantwith a chemical regulator that is capable of inducing expression of thegene or genes which is regulated by the native, non-modified signaltransduction cascade. The plant may further be altered by transformationwith a heterologous gene of interest which is expressed upon treatmentof the plant with the chemical regulator. The method is useful incontrolling or altering traits such as height, shape, development, malesterility, female sterility, and the ability of a plant to withstandcold, salt, heat, drought, disease or pest infestation. The method isespecially useful when constitutive expression of gene(s) involved inmanifestation of these traits might be deleterious to the growth orhealth of the plant. The method has further usefulness in renderingplants capable of functioning as bioreactors for the production ofindustrial or pharmaceutical biomaterials and precursors thereof. In thealternative, the altered plant containing the inactivated signaltransduction cascade can be used in an assay to identifydownstream-acting chemical regulators. That is, the chemical is notdependent upon the signal cascade and is capable of regulating, e.g.,inducing expression of the gene or genes regulated endogenously by thenative, functional cascade.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Partial restriction endonuclease map of lambda tobchrPR1013. The49 kb recombinant phage genome is depicted with the right and left armsof lambda designated. The 19 kb tobacco insert is enlarged below thelambda map. The location of the PR-1a gene (crosshatched box) and thedirection of transcription (arrow) are designates P=Pst, H=HindIII,C=ClaI, R=EcoRI.

FIG. 2. The construction of pBS-PR1013Cla from lambda tobchrPR1013 isshown. The 19 kb DNA fragment between the two ClaI sites was subclonedinto the bluescript plasmid. C=ClaI, LGT agarose=low-gelling temperatwragarose.

FIG. 3. The construction of pBS-PR1013Eco from lambda tobchrPR1013 isdepicted. The 3.6 kb EcoRI fragment containing the PR-1A gene fromlambda tobchrPR1013 is subcloned into Bluescript. R=EcoRI, LGTagarose=low-gelling temperature agarose.

FIG. 4. The construction of pBS-PR1013Eco from pBS-PR1013Cla is shown.The 3.6 kb EcoRI fragment containing the PR-1a gene is subcloned intothe EcoRI site of the bluescript plasmid, C=ClaI, R=EcoRI,LGT=low-gelling temperature agarose.

FIG. 5. The construction of pBS-PR1013Eco Pst from pBS-PR1013Eco isshown. The 600 bp PstI fragment is deleted from pBS-PR1013Eco. P=PstI,R=EcoRI, X=XhoI S=SalI, LGT agarose=low-gelling temperature agarose.

FIG. 6. The construction of pBS-PR1013Eco Pst Xho from pBS-PR1013Eco Pstis shown. The 2 kb XhoI fragment is deleted from the plasmidpBS-PR1013Eco Pst. R=EcoRI, P=PstI, X=XhoI, S=SalI, LGTagarose=low-gelling temperature agarose.

FIG. 7. The construction of pCIB270 from pBI101.3 and pBS-PR1013Eco PstXho is illustrated. The PstI-XhoI fragment containing part of the PR-1agene and 5′ flanking sequence is subcloned into SalI-BamHI digestedpBI101.3. The XhoI and SalI sites are compatible and when ligateddestroy both restriction sites. The PstI site is adapted to a BamHI siteusing a molecular adapter. X=XhoI, P=PstI, (X/S)=site of XhoI and SalIfusion. Neither enzyme will now cut at this (X/S) site. LB=T-DNA leftborder, RB=T-DNA right border. Direction of transcription from the PR-1ainducible region is shown by the arrow on pCIB270. The crosshatched areaon pCIB270 represents the 3′ processing site of the NOS gene. The shadedarea of pCIB270 represents the beta-glucuronidase gene. The stippledarea of pCIB270 represents the neomycin phosphotransferase II gene. Thestriped area of pCIB270 represents the NOS promoter.

FIG. 8. The construction of M13mp18-PR1013Eco Pst Xho andM13mp19-PR1013Eco Pst Xho is shown. The PstI-Asp718 fragment containingpart of the PR-1a coding sequence and the 5′ flanking sequence issubcloned from pBS-PR1013Eco Pst Xho into Asp718-PstI digested M13mp18or 19. R=EcoRI, H=HindIII, P=PstI, K=KpnI (Asp718 is an isoschizomer ofKpnI), LGT agarose=low-gelling temperature agarose.

FIG. 9. Flow diagram depicting the conversion of the ATG codon of PR-1ato a NcoI site. The single stranded DNA of M13mp18-PR1013Eco Pst Xho isshown with a solid line. The sequence TCATGG is converted to CCATGG bysitedirected mutagenesis. K=KpnI, X=XhoI, B=BstEII, P=PstI.

FIG. 10. The construction of pCIB268 is shown. The BstEII-PstI fragmentderived from the replicative form of M13mp18-PR1013Eco Pst Xho.Nco issubcloned into BstEII-PstI digested pBS-PR1013Eco Pst Xho to formpCIB268. X=XhoI, B=BstEII, N=NcoI, P=PstI.

FIG. 11. The construction of pCIB269 from pBS-GUS1.2 and pCIB268 isshown. X=XhoI, N=NcoI, P=PstI. The crosshatched box of pCIB269 depictsthe GUS gene sequences, and the shade box depicts the sequences derivedfrom the PR-1a gene.

FIG. 12. The construction of pBS-GUS1.2 is shown. pBS-GUS1.2 is made viaa three-way ligation from fragments derived from pRAJ265, pBI221.1 andpBluescript. S=SalI, R=EcoRI, N=NcoI.

FIG. 13. The construction of pCIB271 from pCIB269 and pCIB200 is shown.X=XhoI, N=NcoI, R=EcoRI, S=SalI, (S/X)=fusion of SalI and XhoI sites,LGT agarose=low-gelling temperature agarose.

FIG. 14. Restriction endonuclease map of pCIB219. This plasmid isconstructed by adding an EcoRI/XhoI adapter to the pCIB269 XhoI/EcoRIfragment containing the PR-1 and GUS gene and ligating it into SalIrestricted pCIB712.

FIG. 15. Restriction endonuclease imap of pCIB272. This plasmid isconstructed by ligating an Asp718I/BamHI fragment from pCIB282containing a PR-1/GUS gene (−833 to +1 of PR-1a) into Asp718I/BamHIrestricted pCIB200.

FIG. 16. Restriction endonuclease map of pCIB273. This plasmid isconstructed by ligating an Asp718I/BamHI fragment from pCIB283 containnga PR-1/GUS gene (−603 to +1 of PR-1a) into Asp718I/BamHI restrictedpCIB200.

FIG. 17. Restriction endonuclease map of pCIB1004. This plasmid isconstructed by ligating a XhoI/NcoI fragment from pCIB269 (containingthe PR-1a promoter) with the BT gene excised from pCIB10/35Bt(607) as aNcoI/BamHI fragment and SalI/BamHI digested pCIB710.

FIG. 18. Restriction endonuclease map of pCIB200/PR1-BT. This plasmid isconstructed from pCIB1004 and pCIB200.

FIG. 19. Restridction endonuclease map of pCIB1207. A 5.8 kb XbaIfragment of the lambda genomic clone containing the Arabidopsis AHASgene is cloned into XbaI restricted Bluescript.

FIG. 20. Restriction endonuclease map of pCIB1216. A 3.3 kb NcoI/XbaIfragment from pCIB1207 is cloned into pCIB269 which had been restrictedwith NcoI and XbaI to remove the GUS gene.

FIG. 21. Restriction endonuclease map of pCIB1233. A 4.2 kb KpnI/XbaIfragment is isolated from pCIB1216 and ligated into pCIB200 which hadbeen restricted with KpnI and XbaI.

FIG. 22. Restnction endonuclease map of pBSGluc39.1/GUS. A 1462 bpfragment of pBSGluc39.1 is cloned into pBSGUS1.2 which had beenrestricted with NcoI and KpnI.

FIG. 23. Restriction endonuclease map of pCIB200/Gluc39.1-GUS. AKpnI/XbaI fragment containing the β-glucanase promoter and the GUS geneis isolated from pBSGluc39.1/GUS and ligated into pCIB200 restrictedwith KpnI and XbaI.

FIG. 24. Restriction endonuclease map of pCIB200/Gluc39.1-BT. AKpnI/NcoI fragment from pCIB1004 containing the BT gene, a KpnI/NcoIfragment from pBSGluc39.1/GUS, and pCIB200 restricted with KpnI andtreated with calf thymus alkaline phosphatase are ligated.

FIG. 25. Restriction endonuclease map of pBSGluc39.1/AHAS, constructedfrom a NcoI/XbaI fragment of pBSGluc39.1/GUS and a 3.3 kb NcoI/XbaIfragment from pCIB1207 containing the AHAS gene.

FIG. 26. Restriction endonuclease map of pCIB200/Gluc39.1-AHAS. AKpnI/XbaI fragment containing the β-glucanase promoter and the AHAS geneis isolated from pBSGluc39.1/AHAS and ligated into pCIB200 restrictedwith KpnI and XbaI.

FIG. 27. Restriction endonuclease map of pBSGluc39.3/GUS. A 1677 bpfragment of pBSGluc39.3 is cloned into pBS-GUS1.2 which had beenrestricted with NcoI and KpnI.

FIG. 28. Restriction endonuclease map of pCIB200/Gluc39.3-GUS. AKpnI/XbaI fragment containing the β-glucanase promoter and the GUS geneis isolated from pBSGluc39.3/GUS and ligated into pCIB200 restrictedwith KpnI and XbaI.

FIG. 29. Restriction endonuclease map of pCIB200/Gluc39.3-BT. AKpnI/NcoI fragment from pCIB1004 containing the BT gene, a KpnI/NcoIfragment from pBSGluc39.3/GUS, and pCIB200 restricted with KpnI andtreated with calf thymus alkaline phosphatase are ligated.

FIG. 30. Restriction endonuclease map of pBSGluc39.3/AHAS, constructedfrom a NcoI/XbaI fragment of pBSGluc39.3/GUS and a 3.3 kb NcoI/XbaIfragment from pCIB1207 containing the AHAS gene.

FIG. 31. Restriction endonuclease map of pCIB200/Gluc39.3-AHAS. AKpnI/XbaI fragment containing the β-glucanase promoter and the AHAS geneis isolated from pBSGluc39.3/AHAS and ligated into pCIB200 restrictedwith KpnI and XbaI.

FIG. 32. Restriction endonuclease map of pCIB1208. A 5.8 kb XbaIfragment of the lambda genomic clone containing a mutated ArabidopsisAHAS gene is cloned into XbaI restricted Bluescript.

FIG. 33. Restriction endonuclease map of pCIB1230. A 3.3 kb NcoI/XbaIfragment from pCIB1208 is cloned into pCIB269 which had been restrictedwith NcoI and XbaI to remove the GUS gene.

FIG. 34. Restriction endonuclease map of pCIB1232. A 4.2 kb KpnI/XbaIfragment is isolated from pCIB1230 and ligated into pCIB200 which hadbeen restricted with KpnI and XbaI.

FIG. 35. Restriction endonuclease map of pBSGluc39.1/AHAS-SuR,constructed from a NcoI/XbaI fragment of pBSGIuc39.1/GUS and a 3.3 kbNcoI/XbaI fragment from pCIB1208 containing the AHAS gene.

FIG. 36. Restriction endonuclease map of pCIB200/Gluc39.1-AHAS-SuR. AKpnI/XbaI fragment containing the β-glucanase promoter and the AHAS geneis isolated from pBSGluc39.1/AHAS-SuR and ligated into pCIB200restricted with KpnI and XbaI.

FIG. 37. Restriction endonuclease map of pBSGluc39.3/AHAS-SuR,constructed from a NcoI/XbaI fragment of pBSGluc39.31GUS and a 3.3 kbNcoI/XbaI fragment from pCIB1208 containing the AHAS gene.

FIG. 38. Restriction endonuclease map of pCIB200/Gluc39.3-AHAS-SuR. AKpnI/XbaI fragment containing the β-glucanase promoter and the AHAS geneis isolated from pBSGluc39.3/AHAS-SuR and ligated into pCIB200restricted with KpnI and XbaI.

FIG. 39. A DNA sequence comparison of five SAR8.2 cDNAs; SAR8.2a, b, c,d and e. Bases identical to the SAR8.2a sequence are represented by adot. Mismatches are represented by a capital letter. Gaps arerepresented by dashes. The length of each cDNA is given in brackets atthe 3′ end of the sequence. Alignments were done using the MacVector(IBI) software, with larger gaps being determined by visual inspection.

FIG. 40. An amino acid sequence comparison of the five SAR8.2 cDNA openreading frames. Amino acids identical to the SAR8.2a sequence arerepresented by a dot. Mismatches are given by capital letters, and gapsare represented by dashes. An asterisk indicates a stop codon. TheSAR8.2e sequence is continued on the next line to show its alignmentwith homologous segments of the other proteins and itself.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID No. 1: The genornic DNA sequence of the 2 kb pair fragmentbetween the XhoI and BglII sites of the tobacco PR-1a gene. Thetranscription start site occurs at about nucleotide 903. The polypeptidesequence encoded by the coding portion of the gene is disclosed in SEQID No. 45.

SEQ ID No. 2: The genomic DNA sequence of the tobacco PR-1′ gene. Theamino acid sequence of the polypeptide which is coded by the codingregion of this gene is disclosed in SEQ ID No. 46.

SEQ ID No. 3: The cDNA sequence encoding a cucumber chitinase/lysozymeprotein cloned into the plasmid pBScucchi/chitinase.

SEQ ID No. 4: The cDNA sequence encoding a PR-R major protein clonedinto the plasmid pBSPRR-401.

SEQ ID No. 5: The genomic DNA sequence of the tobacco basic β-1,3glucanase gene contained in the clone pBSGluc39.1.

SEQ ID NO. 6: The genomic DNA sequence of the tobacco basicβ-1,3-glucanase gene contained in the clone pBSGluc39.3.

SEQ ID No. 7: The cDNA sequence encoding a PR-Q protein cloned into theplasmid pBScht15.

SEQ ID No. 8: The DNA sequence of an isolated cDNA contained in theplasmid pBSGL6e. The cDNA encodes the acidic form of β-1,3-glucanase.

SEQ ID No. 9: The cDNA sequence encoding a PR-1a protein cloned into theplasmid pBSPR1-207.

SEQ ID No. 10: The cDNA sequence encoding a PR-1b protein cloned intothe plasmid pBSPR1-1023.

SEQ ID No. 11: The cDNA sequence encoding a PR-1c protein cloned intothe plasmid pBSPR1-312.

SEQ ID NO. 12: The cDNA sequence encoding a PR-P protein cloned into theplasmid pBScht28.

SEQ ID No. 13: A partial cDNA sequence encoding a PR-O′ protein clonedinto the plasmid pBSGL6e.

SEQ ID No. 14: The full length cDNA sequence encoding a PR-O′ proteincloned into the plasmid pBSGL5B-12.

SEQ ID No. 15: The cDNA sequence encoding a SAR8.2a protein cloned intothe plasmid pCIB/SAR8.2a.

SEQ ID No. 16: The cDNA sequence encoding a SAR8.2b protein cloned intothe plasmid pCIB/SAR8.2b.

SEQ ID No. 17: The cDNA sequence encoding a SAR8.2c protein.

SEQ ID No. 18: The cDNA sequence encoding a SAR8.2d protein.

SEQ ID No. 19: The cDNA sequence encoding a SAR 8.2e protein.

SEQ ID No. 20: The cDNA sequence encoding a basic β-1,3-glucanaseprotein cloned into the plasmid pGLN17. The encoded amino acid sequenceis provided in SEQ ID No. 98.

SEQ ID No. 21: The cDNA sequence encoding a PR-2 protein cloned into theplasmid pBSGL117.

SEQ ID No. 22: The cDNA sequence encoding a basic cucumber peroxidaseprotein cloned into the plasmid pBSPER1.

SEQ ID No. 23: The cDNA sequence encoding a PR-O protein cloned into theplasmid pBSGL134.

SEQ ID No. 24: The cDNA sequence encoding a PR-N protein cloned into theplasmid pBSGL167.

SEQ ID No. 25: The cDNA sequence encoding a PR-O-related tobaccoβ-1,3-glucanase protein, designated PR-2′, cloned into the phage lambdatobcDNAGL161.

SEQ ID No. 26: The cDNA sequence encoding a PR-O-related tobaccoβ1,3-glucanase protein, designated PR-2″, cloned into the phage lambdatobcDNAGL153.

SEQ ID No. 27: The cDNA sequence encoding a cucumber peroxidase proteincloned into the plasmid pBSPERB24.

SEQ ID No. 28: The cDNA sequence encoding another cucumber peroxidaseprotein cloned into the plasmid pBSPERB25.

SEQ ID No. 29: The cDNA sequence encoding a basic tobaccochitinase/lysozyme protein cloned into the plasmid pBSCL2.

SEQ ID No. 30: The cDNA sequence encoding an acidic tobaccochitinase/lysozyme protein cloned into the plasmid pBSTCL226.

SEQ ID No. 31: The cDNA sequence encoding a PR-4a protein cloned intothe plasmid pBSPR-4a.

SEQ ID No. 32: The cDNA sequence encoding a PR-4b protein cloned intothe plasmid pBSPR-4b.

SEQ ID No. 33: The cDNA sequence encoding an Arabidopsis PR-1 proteincloned into plasmid pAPR1C-1.

SEQ ID No. 34: The cDNA sequence encoding an Arabidopsis PR-4 proteincloned into pSLP-1.

SEQ ID No. 35: The cDNA sequence encoding an Arabidopsis PR-R proteincloned into pATL12a.

SEQ ID No. 36: The full sequence of a cucumber chinnase EcoR1 genomicclone.

SEQ ID No. 37: The cDNA sequence encoding an Arabidopsis class IVchitinase with a hevein domain (pChit4-TA).

SEQ ID No. 38: The cDNA sequence encoding an Arabidopsis class IVchitinase without a hevein domain (Chit4-TB).

SEQ ID No. 39: The cDNA sequence of the wheat gene WCI-1.

SEQ ID No. 40: The partial cDNA sequence of the 5′ end of the wheat geneWCI-2 which encodes a lipoxygenase isozyme. The partial sequence of the3′ end of this cDNA is provided in SEQ ID No. 41.

SEQ ID No. 41: The partial cDNA sequence of the 3′ end of the wheat geneWCI-2 which encodes a lipoxygenase isozyme. The partial sequence of the5′ end of this cDNA is provided in SEQ ID No. 40.

SEQ ID No. 42: The cDNA sequence of the wheat gene WCI-3.

SEQ ID No. 43: The cDNA sequence of the wheat gene WCI-4.

SEQ ID No. 44: The cDNA sequence of the wheat gene WCI-5.

SEQ ID No. 45: The amino acid sequence encoded by the coding portion ofSEQ ID No. 1.

SEQ ID No. 46: The amino acid sequence encoded by the coding portion ofSEQ ID No. 2.

SEQ ID No. 47: A representative molecular adaptor sequence.

SEQ ID NO. 48: Oligonucleotide primer for the PR-1 gene.

SEQ ID NO. 49: Oligonucleotide primer for GUS gene.

SEQ ID NO. 50: Oligonucleotide primer for the AHAS gene.

SEQ ID NO. 51: Oligonucleotide primer for the BT gene.

SEQ ID No. 52: Amino acid sequence of PR-R Major.

SEQ ID No. 53: Amino acid sequence of PR-R Minor.

SEQ ID No. 54: The Pst/BamHI olignucleotide adaptor used in Example 25.

SEQ ID No. 55: The oligonucleotide primer used in Example 27.

SEQ ID No. 56: The oligonucleodde pimer used in Example 30.

SEQ ID Nos. 57-58: Oligonucleotides used in Example 35.

SEQ ID Nos. 59-60: Oligonucleotides used in Example 36.

SEQ ID Nos. 61-62: Oligonucleotides used in Example 42.

SEQ ID No. 63: Oligonucleotide used in Example 44.

SEQ ID No. 64: Oligonucleotide used in Example 45.

SEQ ID Nos. 65-67: Oligonucleotides used in Example 46.

SEQ ID Nos. 68-69: Oligonucleotides used in Example 48.

SEQ ID No. 70: Oligonucleotide used in Example 49.

SEQ ID Nos. 71-72: Oligonucleotides used in Example 51.

SEQ ID No. 73: Oligonuclootide used in Example 53.

SEQ ID No. 74: Oligonucleotide used in Example 55.

SEQ ID No. 75: Amino acid sequence recited in Example 55.

SEQ ID Nos. 76,78, 80: Oligonucleotides used in Example 58.

SEQ ID Nos. 77, 79, 81: Amino acid sequence recited in Example 58.

SEQ ID Nos. 82-83: Oligonucleotides used in Example 62.

SEQ ID Nos. 84-85: Oligonucleotides used in Example 72.

SEQ ID Nos. 86-89: Oligonucleotides used in Exmle 73.

SEQ ID Nos. 90-91: Oligonucleotides used in Example 79.

SEQ ID No. 92: Nucleotide sequence recited in Example 80.

SEQ ID Nos. 93-96: Oligonucleoddes rcied in Example 81.

SEQ ID No. 97: Oligonucleotide used in Example 84.

SEQ ID No. 98: Amino Acid sequence encoded by the coding sequence withinSEQ ID No. 20.

SEQ ID No. 99: A tobacco protein-synthesis independent gene involved inthe regulation of the systemic acquired resistance response designatedp1.1.1.

SEQ ID No. 100: A tobacco protein-synthesis independent gene involved inthe regulation at of the systemic acquired resistance responsedesignated p11.3.8.

SEQ ID No. 101: The 5′ DNA sequence of a tobacco protein-synthesisindependent gene involved in the regulation of the systemic acquiredresistance response designated p11.30.13.

SEQ ID No. 102: The DNA sequence of the 3′ end of the sameprotein-synthesis independent gene described in Seq. ID. No. (iii)cloned from tobacco and involved in the regulaton of the systemicacquired resistance response designated p11.30.13. This sequence isderived from the non-coding strand (i.e. the “bottom” strand). The firstbase listed is therefore located in the furthest 3′ position.

SEQ ID No. 103: A tobacco protein-synthesis independent gene involved inthe regulation of the systemic acquired resistance response designatedp1.4.3. This sequence is identical to the thiozedoxin gene published byBrugidou et al., Mol. Gen. Genet. 238: 285-293 (1993).

SEQ ID No. 104: A protein-synthesis dependent SAR gene cloned fromtobacco designated p66B1.

SEQ ID No. 105: A protein-synthesis dependent SAR gene cloned fromtobacco designated p14.22.3.

SEQ ID No. 106: An Arabidopsis protein-synthesis independent geneinvolved in the regulation of the systemic acquired resistance responsedesignated pDPA2.

SEQ ID No. 107: Amino Acid sequence of an SAR8.2a protein.

SEQ ID No. 108 Amino Acid sequence of an SAR8.2b protein.

SEQ ID No. 109 Amino Acid sequence of an SAR8.2c protein.

SEQ ID No. 110 Amino Acid sequence of an SAR8.2d protein.

SEQ ID No. 111 Amino Acid sequence of an SAR8.2e protein.

DETAILED DESCRIPTION OF THE INVENTION A. DEFINITIONS

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided:

Anti-pathogenic Sequence: A DNA molecule encoding a plantpathogenesis-related (PR) protein, or a DNA molecule with substantialhomology thereto, which is capable of conferring enhanced resistance ortolerance to disease and/or pests when expressed in a plant, seed, orplant tissue.

Anti-sense Mechanism: A mechanism for regulation of gene expressionbased on the presence in a cell of a RNA molecule complementary to atleast a portion of the mRNA encoded by the gene. This mechanism isthought to involve controlling the rate of translation of mRNA toprotein due to the presence in a cell of an RNA molecule complementaryto at least a portion of the mRNA being translated.

Associated DNA Sgguence: A DNA sequence whose cellular activity either(1) regulates the activity of another DNA sequence or (2) is regulatedby another DNA sequence. This definition specifically embraces, but isnot limited to, sequences which are physically adjacent in a continuousDNA strand or which are physically separated. Physical separationincludes, for example, separation within the same DNA strand, locationwithin different DNA strands, or discontinuous interspered sequences(eg., alternating regulatable and coding sequences) in one strand.

Chemically Regulatable DNA Scuence: A DNA sequence which is capable ofregulating the transcription of an associated DNA sequence where theregulation is dependent on a chemical regulator. The sequences may be ofnatural or synthetic origin.

Chemically Regulatable Gene: A gene containing at least one non-codingchemically regulatable DNA sequence and at least one associated codingDNA sequence. The genes may be of natural, synthetic or partiallynatural/partially synthetic origin.

Chemical Regulator (for a chemically regulatable DNA sequence): Anelemental or molecular species which controls (e.g., initiates,terminates, increases or reduces), by direct or indirect action, theactivity of a chemically regulatable DNA sequence in a system in whichthe chemical regulator is not normally found in an active form in anamount sufficient to effect regulaton of transcription, to the degreeand at the time desired, of a transcribable DNA sequence associated withthe chemically regulatable DNA sequence. This terminology is intended toembrace situations in which no or very little regulator is present atthe time transcription is desired or in which some regulator is presentbut increased or decreased regulation is required to effect more or lesstranscription as desired.

Thus, if the system containing the chemically regulatable DNA sequenceis a plant, for example a transgenic plant, a chemical regulator is aspeces not naturally found in the plant in an amount sufficient toeffect chemical regulation, and thus transcription of an associatedgene, to the desired degree at the time desired.

By “direct action” it is intended that the chemical regulator actionresult from the direct interaction between the chemical regulator andthe DNA sequence. By “indirect action” it is meant that the regulatoraction results from the direct interaction between the chemicalregulator and some other endogenous or exogenous component in thesystem, the ultimate result of that direct interaction being activationor suppression of the activity of the DNA sequence. By “active form” itis intended that the chemical regulator be in a form required to effectcontrol.

Chimeric Sequence or Gene: A DNA sequence containing at least twoheterologous parts, e.g., parts derived from naturally occuring DNAsequences which are not associated in their naturally occurring states,or containing at least one part that is of synthetic origin and notfound in nature.

Coding DNA Sequence: A DNA sequence which, when transcribed andtranslated, results in the formation of a cellular polypeptide.

Constitutive transcription: Transcription of substantially fixed amountsof a DNA sequence, irrespective of environmental conditions.

Gene: A discrete chromosomal region which is responsible for a discretecellular product.

Inducers: Molecules that cause the production of larger amounts ofmacromolecules, compared to the amounts found in the absence of theinducer.

Inducible Protein: Proteins whose rate of production can be increased bythe presence of inducers in the environment.

Non-coding DNA Sequence: A DNA sequence, which is not transcribed andtranslated, resulting in the formation of a cellular polypeptide whenassociated with a particular coding DNA sequence. A sequence that isnon-coding when associated with one coding sequence may actually becoding when associated with another coding or non-coding sequence.

Phenotypic Trait: An observable property resulting from expression of agene.

Plant Tissue: Any tissue of a plant in planta or in culture. This termincludes, but is not limited to, whole plants, plant cells, plantorgans, plant seeds, protoplasts, callus, cell cultures and any groupsof plant cells organized into structural and/or functonal units. Theuse, of this term in conjunction with, or in the absence of, anyspecific type of plant tissue as listed above or otherwise embraced bythis definition is not intended to be exclusive of any other type ofplant tissue.

PR, or Pathogenesis-Related Proteins: Proteins expressed in plantsreacting hypersensitively towards pathogens. This term embraces, but isnot limited to, SAR8.2a and SAR8.2b proteins, the acidic and basic formsof tobacco PR-1a, PR-1b, PR-1c, PR-1′, PR-2, PR-2′, PR-2″, PR-N, PR-O,PR-O′, PR-4, PR-P, PR-Q, PR-S, and PR-R major proteins, cucumberperoxidases, basic cucumber peroxidase, the chitinase which is a basiccounterpart of PR-P or PR-Q, and the beta-1,3-glucanase (glucanendo-1,3-β-glucosidase, EC 3.2.1.39) which is a basic counterpart ofPR-2, PR-N or PR-O, the pathogen-inducible chitinase from cucumber,class IV chitinases with and without a hevein domain, and the WCI(“Wheat Chemically Induced”) gene proteins from wheat. A hypersensitivereaction is characterized by a local necrosis of the tissues immediatelysurrounding the infection site of the pathogen and a subsequentlocalization of the pathogen, which is in contrast to a sensitivereaction wherein the pathogen spreads throughout the plant. Pathogensare, for example, viruses or viroids, e.g. tobacco or cucumber mosaicvirus, ringspot virus or necrosis virus, pelargonium leaf curl virus,red clover mottle virus, tomato bushy stunt virus, and like viruses,fungi, e.g. Phythophthora parasitica or Peronospora tabacina, bacteria,e.g. Pseudomonas syringae or Pseudomonas tabac, or aphids, e.g. Myzuspersicae. This list is not limiting in any respect.

Regulation: The increasing (inducing) or decreasing (repressing) of thelevel of expression of a gene or the level of transcription of a DNAsequence. The definition is not intended to embrace any particularmechanism.

Substantially Pure DNA Sequence: A DNA molecule (sequence) isolated insubstantially pure form from a natural or non-natural source. Such amolecule may occur in a natural system, for example, in bacteria,viruses or in plant or animal cells, or may be provided, for example, bysynthetic means or as a cDNA. Substantially pure DNA sequences aretypically isolated in the context of a cloning vector. Substantiallypure means that DNA sequences other than the ones intended are presentonly in marginal amounts, for example less than 5%, less than 1%, orpreferably less than 0.1%. Substantially pure DNA sequences and vectorscontaining may be, and typically are, provided in solution, for examplein aqueous solution containing buffers or in the usual culture media.

Substantial Sequence Homology: Substantial sequence homology means closestructural relationship between sequences of nucleotides or amino acids.For example, substantially homologous DNA sequences may be 80%homologous, preferably 90% or 95% homologous, and substantiallyhomologous amino acid sequences may typically be 50% homologous, ormore. Homology also includes a relationship wherein one or severalsubsequences of nucleotides or amino acids are missing, or subsequenceswith additional nucleotides or amino acids are interdispersed.

B. ABBREVIATIONS

The following abbreviations are used herein:

ATCC American Type Culture Collection ATP adenosine triphosphate bp basepair BT Bacillus thuringiensis endotoxin CAT chloramphenicolacetyltransferase CETAB hexadecyltrimethylammonium bromide 2,4-D2,4-dichlorophenoxyacetic acid DTT dithiothreitol dicamba3,6-dichloro-2-methoxybenzoic acid EDTA ethylendiamineN,N,N′,N′-tetraacetic acid GUS beta-1,3-glucuronidase kb kilo base pairLUX luciferase MES 2-(N-morpholino)ethanesulfonic acid MU 4-methylumbelliferyl glucuronide NOS nopaline synthase NPT neomycinphosphotransferase NRRC designation for deposits made with theAgricultural Research Culture Collection, International DepositingAuthority, 1815 N. University Street, Peoria, Illinois 61604 OCSoctopine synthase PEG polyethylene glycol picloram4-amino-3,5,6-trichloropicolinic acid PR protein Pathogenesis-relatedprotein SAR Systemic Acquired Resistance SDS sodium dodecyl sulfate TFAtrifluoroacetic acid TMV tobacco mosaic virus Tris-HCltris(hydroxymethyl)methylamine hydrochloride WCI Wheat ChemicallyInduccd (gene nomenclature designation)

C. Introduction

The present invention describes the identification, isolation, andcloning of DNA sequences which are capable of regulating thetranscription in plant tissue of an associated DNA sequence where theregulation is dependent upon a chemical regulator. These DNA sequencescan be utilized to construct chimeric genes in which the expression ofthe gene can be regulated by such regulators. The ability to regulatethe expression of a chimeric gene in a transgenic plant by a chemicalmethod is useful to obtain suitable expression of the phenotypic traitwith minimal adverse effect on the growth and development of the plant.This regulation is important in the production of secondary products orother cloned products in plant tissue in culture or bioreactors. Theregulation of the cloned sequence is also important in the regulation ofother gene products by an anti-sense mechanism.

The expression of a given coding sequence at any specific time can beregulated by use of a chemical regulator, typically applied to the planttissue. Genes involved in the control of distinct developmentaltransition stages can also be regulated by associating a chemicallyregulatable DNA sequence with an appropriate coding DNA sequence. Inthis manner the development of a plant can be halted at a specific stageuntil, or accelerated at a specific rate when, the level of a chemicalregulator is increased or reduced.

The chemically regulatable DNA sequences can be utilized to drive theexpression of foreign genes which, for example, confer herbicideresistance or tolerance (e.g., atrazine tolerance in soybean), conferinsect resistance (e.g., Bacillus thuringienis crystal protein incotton) or require selective expression (such as male or femalesterility). The chemically regulatable DNA sequences can also beutilized to drive the transcription of a DNA sequence which will controlthe expression of a second coding sequence by an anti-sense mechanism.

The present invention further provides a) anti-pathogenic sequencesderived from novel cDNA clones coding for plant pathogenesis-relatedproteins; b) chimeric DNA constructions useful for producing transgenicdisease-resistant plants which comprise a first DNA sequence whichpromotes in a plant the constitutive transcription of the second DNAsequence, and a second DNA sequence which is a coding sequence of aninducible plant pathogenesis-related protein or a coding sequence havingsubstantial sequence homology to a coding sequence of an inducible plantpathogenesis-related protein; c) vectors containing such chimeric DNAconstructions; and c) transgenic plants, transgenic plant tissue,propagules and seeds of transgenic plants containing the chimeric DNAconstructions for producing disease-resistant plants.

Also provided is a novel method for differential screening and enrichingcDNA populations comprising a) providing single-stranded cDNA frominduced and uninduced cDNA populations, the single-stranded cDNA fromthe induced and uninduced populations having opposite DNA polarity, andthe cDNA from the uninduced population having a biotin-affinity tag; b)hybridizing the single-stranded cDNA populations of step a) with eachother, and c) separating the hybridized mixture of step b) bybiotin-avidin chromatography to enrich for single-stranded cDNAs fromthe induced population which are not hybridized to the cDNA from theuninduced population.

Further provided herein is a method for cloning cDNA's encodingdisease-resistance proteins comprising a) providing tissue induced tosystemic acquired resistance or localized acquired resistance usingbiological inducers or chemical inducers, and b) isolating cDNA clonesincluding disease-resistance proteins. Although cDNA's have beenproduced previously from RNA isolated from pathogen-infected material,this is the first example of producing cDNA's from RNA isolated fromuninfected portions of the plant that have been induced to resistance bypathogens. That is, the tissue is uninfected by pathogens, but isdemonstrating acquired resistance. This method also encompasses the useof chemical inducers.

The production of transgenic plants which are disease resistant can nowbe realized by the present invention which is directed to, among otherthings, chimeric DNA constructions useful for producing transgenicdisease-resistant plants. The chimeric DNA constructions contain acoding DNA sequence which encodes a plant pathogenesis-related proteinwhich is normally pathogen-induced in a wild type plant, and a promoterDNA sequence which provides for the constitutive expression ofpathogenesis-related proteins or anti-sense mRNA for PR-proteins in atransgenic plant containing the chimeric DNA construction.

Accordingly, the present invention includes, but is not limited to, thefollowing:

a. A chimeric gene whose expression in plant tissue is regulated by achemical regulator.

b. A substantially pure chemically regulatable DNA sequences capable ofcontrolling genetic activity in plant tissue of other DNA sequences inresponse to a chemical regulator.

c. Chemically regulatable DNA sequences in combination with part but notall of any coding DNA sequence with which they are associate innaturally occurring genes.

d. Vectors containing the chemically regulatable DNA sequences with orwithout other parts of naturally occurring genes in which they mayoccur.

e. Vectors containing the chimeric genes of (a).

f. Plant tissue, plants and seeds derived from cells transformed bythese vectors.

g. A process for chemically regulating the expression of DNA codingsequences associated with the chemically regulatable DNA sequences, forexample to regulate a phenotypic trait.

h. A process for identifyig new chemical regulators using chimeric genesof this invention and plant tissue containing them.

i. Signal peptide sequences in proteins encoded by chemically regulatedgenes.

j. Substantially pure pathogenesis-related protein genes and cDNAs.

k. Transgenic plants constitutively expressing pathogenesis-relatedprotein genes providing an enhanced disease-resistant phenotype withrespect to wild-type plants.

l. Transgenic plants constitutively transcribing sense or anti-sensemRNA strands of DNA sequences encoding plant pathogenesis-relatedproteins, or transcribing sense or anti-sense mRNA strands of DNAsequences substantially homologous to genomic or cDNA sequences encodingplant pathogenesis-related proteins, such transgenic plants thus havingan enhanced disease-resistant phenotype with respect to wild-typeplants.

Other aspects of the present invention are discernible from thefollowing description.

D. CHEMICALLY REGULATABIE DNA SEQUENCES

The present invention is concerned with non-coding DNA sequences whichare capable of regulating, under the influence of a chemical regulator,the trascription of an associated DNA sequence in a plant or planttissue. Specifically, the present invention embraces a non-coding DNAsequence capable of regulating the transcription of an associated DNAsequence in a plant or plant tissue wherin this regulation is dependentupon a chemical regulator. Preferably the DNA sequences exist insubstantially pure form relative to the gene and genome in which theyoccur if they come from a natural source, or relative to a DNA mixture,if they occur in a synthetic mixture.

Preferably the non-coding chemically regulatable DNA sequences of thisinvention are those which, when associated with a coding DNA sequence,regulate the expression of the coding sequence in plant tissue, theextent of regulation being dependent upon a chemical regulator. Suchsequences can be synthesized de novo or derived (for example, isolatedor cloned) from a naturally occurring chemically regulatable gene. Theoccurrence of chemically regulatable DNA sequences of the invention isnot limited to plant tissue; i.e., the chemically regulatable DNAsequences may be derived from a variety of natural sources, e.g.,bacterial, viral or animal sources. It may be isolated, for example,from the 5′ flanking region or from the 3′ flankig region of a naturallyoccurring chemically regulatable gene. Alternatively, the non-coding DNAsequence may be chemically synthesized or enzymatically synthesized ascDNA identical to, or having substantial sequence homology to, theisolated sequence. By cloning the chemically regulatable, non-coding DNAsequence, the sequence can be separated from other sequences which areadjacent to it in the naturally occurring gene. In this manner asubstantially pure DNA sequence can be obtained.

In the context of the present invention, regulation embraces chemicalregulators which are either inducing or repressing regulators. Examplesof chemically repressible genes, that is, genes which are repressed by arepressing chemical, include, for example, genes like TrpR or AroH wherethe addition of the tryptophan repressor or tryptophan itself repressesthe expression from these genes. Many other genes exist that areregulated by this type of end-product repression and in each case theend-product acts as a chemical regulator that can be used to repress theexpression of the gene. The present invention embraces the regulatablesections of such genes by themselves or as part of chimericconstructions which can be chemically regulated for transcription of anassociated DNA sequence in a plant or plant tissue.

Examples of chemically inducible genes, that is, genes which are inducedby an inducing chemical regulator, are the PR protein genes, especiallythe tobacco PR protein genes, for example the PR-1a, PR-1b, PR-1c,PR-1′, PR-Q, PR-R and PR-S genes, the cucumber chitinase gene, and thebasic and acidic tobacco β-1,3-glucanase genes. In a particular aspectthe present invention comprises a substantially pure DNA sequence whichis, or has substantial sequence homology to, a non-coding chemicallyregulatable DNA sequence which is part of a naturally occurringchemically regulatable gene, for example of a naturally occurringchemically regulatable gene in a plant or plant tissue from a monocot,dicot or gymnosperm. Preferably such a DNA sequence is capable ofregulating the transcription of an associated DNA sequence in a plant orplant tissue wherein said associated DNA sequence is a coding sequence.The transcription of said DNA sequence may be regulated by a repressingchemical regulator or an inducing chemical regulator. DNA sequencesparticularly considered are those wherein the chemically regulatablegene is a PR protein gene, for example from a dicotyledonous plant, e.g.tobacco or cucumber. Most preferred are DNA sequences wherein thechemically regulatable gene is a tobacco PR-1a, PR-1b, PR-1c, PR-1′,PR-Q or PR-R gene, a cucumber chitinase gene, or a basic or acidicβ-1,3-glucanase gene, in particulr the tobacco PR-1a and PR-1′ gene, butalso the cucumber chitinase gene and the basic and acidic tobaccoβ-1,3-glucanase genes. Foremost considered are DNA sequences wherein thechemically regulatable gene is a tobacco PR-1a or basic β-1,3-glucanasegene.

The chemically inducible DNA sequences of the preferred PR and relatedgenes of the invention apparently occur in the non-coding sequences ofthe adjacent region 5′ flanking to the coding sequences. As arepresentative example, in a sequence isolated from an approximately6500 base pair fragment of the tobacco PR-1a gene containing part of thecoding region, the region with about 900 to about 1200 base pairs,naturally adjacent to the transcriptional start site, has been found tobe chemically inducible. Inducibility is retained in a fragment thereofcontaining about 500 to about 700 base pairs naturally adjacent to thetranscriptional start site. Most preferred are therefore DNA sequenceswhich are located in the 5′ flankig region of said PR and related genes,for example in the 1200 base pairs adjacent to the transcriptional startsite.

The invention further embraces a process for the preparation of anon-coding DNA sequence capable of regulating the transcription of anassociated DNA sequence in a plant or plant tissue wherein thisregulation is dependent upon a chemical regulator, and of DNA sequenceshaving substantial homology to said non-coding sequences, characterizedin that the DNA is isolated from a naturally occurring gene insubstantially pure form or is synthesized chemically or enzymatically.

In particular, the invention embraces a process for the preparation of asubstantially pure chemically regulatable DNA sequence from a chemicallyregulatable gene in a naturally occurring system containing that gene,which process comprises the steps of

(a) activating expression in said system of RNA from the chemicallyregulatable gene;

(b) isolating said RNA;

(c) differentially screening a genomic library for clones correspondingto RNA isolated from said activated system that is less abundant orabsent in RNA isolated from a control system that is not activated;

(d) isolating a genomic clone;

(e) subcloning the chemically regulatable gene from said genomic clone;and

(f) isolating the desired chemically regulatable DNA sequence.

Moreover the invention embraces such a process wherein said system is aplant, which process comprises the steps of:

(a) activating expression in said plant of polyA+ RNA from thechemically regulatable gene;

(b) isolating said polyA+ RNA;

(c) constructing a cDNA library from said polyA+ RNA;

(d) differentially screening said cDNA library with cDNA generated fromRNA in a control plant that is not activated;

(e) isolating cDNA clones that are chemically regulatable from thepopulation of clones in (d) that do not correspond to cDNA clonesgenerated from RNA in a control plant that is not activated;

(f) isolating a genomic clone from a genomic library of said plant usingas a probe the cDNA clone of step (e);

(g) subcloning the chemically regulatable gene from said genomic clone;and

(h) isolating the desired chemically regulatable DNA sequence.

Preferred is such a process wherein said chemically regulatable gene isa chemically inducible gene, for example a PR protein gene. Furtherpreferred is such a process wherein said PR protein gene is activated instep (a) by a chemical inducer or a pathogen.

The invention further embraces substantially pure DNA prepared by thementioned processes.

E. CHEMICALLY REGULATABLE DNA SEQUENCES WITH PARTS OF NATURALLYOCCURRING CODING SEQUENCES

In addition to the entirely non-coding chemically regulatable DNAsequences described above in Part D, this invention also provides forthe noncoding DNA sequence of a naturally occurring chemicallyregulatable DNA sequence in combination with part but not all of acoding sequence with which the regulatable sequence is associated in anaturally occurrng gene. More specifically, the present inventionembraces, preferably in substantially pure form, a DNA sequence whichcomprises a first DNA component sequence which is, or has substantialsequence homology to, a non-coding chemically regulatable DNA sequenceof a naturally occurring chemically regulatable gene, this firstcomponent sequence being capable of regulating the transcription of anassociated DNA sequence in a plant or plant tissue, wherein thisregulation is dependent upon a chemical regulator, and a second DNAcomponent sequence which is, or has substantial sequence homology to,part but not all of a transcrnbable DNA sequence with which the firstcomponent is associated in the naturally occurring chemicallyregulatable gene. The naturally occurring chemically regulatable genemay be of plant origin, for example occurring in a monocotyledonous ordicotyledonous plant, and may be regulated by a repressing or aninducing chemical regulator. The second DNA component sequence willtypically be a coding sequence. A preferred second DNA sequence for thepresent invention is a sequence which codes for the signal peptide ofany protein expressed by the naturally occurring chemically regulatablegene.

In particular the invention embraces such a DNA sequence comprising afirst non-coding chemically regulatable DNA sequence and a second codingDNA sequence from a PR protein gene, for example a PR protein gene froma dicotyledenous or monocotyledenous plant. Preferred is a DNA sequencefrom a PR protein gene wherein the transcription is regulated by aninducing chemical regulator. In particular the invention embraces a DNAsequence wherein the first non-coding DNA sequence is located in the 5′flanking region of one of the PR protein genes, for example located inthe approximately 2000 base pairs adjacent to the transcriptional startsite of the PR protein gene. Most preferred is a DNA sequence comprisinga preferred first non-coding sequence as mentioned above and a secondcoding DNA sequence coding for a signal peptide as mentioned above.

Most preferred are substantially pure DNA sequences as shown in SEQ IDNos. 1, 2, 5 and 6, and substantially pure DNA sequences havingsubstantial sequence homology to the sequences shown in any one of thesefigures. These sequences are examples of chemically regulatable DNAsequences comprising a first non-coding and a second coding DNA sequenceand are derived from the PR protein genes tobacco PR-1a, PR-1′, and fromtwo forms of basic tobacco β-1,3-glucanase, respectively. SEQ ID No. 1shows the sequence of the representative PR-1a gene from tobacco.Nucleotides 1 to about 1150 are the non-coding, 5′ flanking chemicallyinducible DNA sequence and part of the PR-1a coding sequence which isnaturally occurring in a tobacco plant. In this case the chemicallyinducible component sequence is adjacent to the coding sequence. Thosenucleotides which code for the first thirty amino acids constitute thecoding sequence for the signal peptide of the PR-1a protein. The codingsequence can be removed from the non-coding sequence to generate thenon-coding, chemically inducible DNA sequence free of any codingsequence described above in Part A. Such removal can be accomplished byconventional techniques, such as restriction enzyme digestions.

The invention further embraces a method for the preparation of a DNAsequence which comprises a first DNA component sequence which is, or hassubstantial sequence homology to, a non-coding chemically regulatableDNA sequence of a naturally occurring chemically regulatable gene, thisfirst component sequence being capable of regulating the transcriptionof an associated DNA sequence in a plant or plant tissue, wherein thisregulation is dependent upon a chemical regulator, and a second DNAcomponent sequence which is, or has substantial sequence homology to,part but not all of a transcribable DNA sequence with all which thefirst component is associated in the naturally occurring chemicallyregulatable gene, characterized in that the DNA is isolated from anaturally occurring gene in substantially pure form or is synthesizedchemically or enzymatically. Preferred are the particular processesmentioned in Part D above and the substantially pure DNA sequencesobtained thereby.

F. CHIMERIC GENES CONTAINING CHEMICALLY REGULATABLE DNA SEQUENCES

A further aspect of the present invention is a chimeric DNA sequence(chimeric gene) containing at least one chemically regulatable DNAsequence. Two types of such chimeric sequences are provided as examples.The simpler, or two-part tppe chimeric DNA sequence comprises achemically regulatable DNA sequence and a transcribable DNA sequencesuch that the chimeric gene is capable of being expressed in planttissue under the proper conditions of chemical regulation. Morespecifically, the invention embraces a chemically regulatable chimericDNA sequence comprising a first non-coding, chemically regulatable DNAcomponent sequence which is capable of regulating the transcription ofan associated DNA sequence in a plant or plant tissue, wherein thisregulation is dependent upon a chemical regulator, and a second DNAcomponent sequence capable of being transcribed in a plant or planttissue. The DNA component sequences may be derived from natural sourcesor be prepared synthetically.

The second DNA sequence may be transcribed as an RNA which is capable ofregulating the expression of a phenotypic trait by an anti-sensemechanism. Alternatively, the second DNA sequence in the chimeric DNAsequence may be transcribed and translated, i.e. coded, in the planttissue to produce a polypeptide resulting in a phenotypic trait. Thechimeric gene is constructed such that the second DNA sequence isproperly associated with, typically in an adjacent orientation to, thechemically regulatable DNA sequence to ensure transcription. Associationis effected with conventional techniques.

Preferred is a chimeric DNA sequence comprising a first non-codingchemically regulatable DNA component and a second transcribable DNAcomponent wherein the first non-coding DNA sequence is one of thepreferred DNA sequences mentioned above under Part D. The first DNAcomponent sequence may be regulated by a repressing or by an inducingchemical regulator. A particular sequence of the invention is a chimericDNA sequence wherein the first DNA component sequence has substantialsequence homology to a chemically regulatable DNA sequence in anaturally occurring chemically regulatable gene from a plant.

Preferred is a chimeric DNA sequence wherein the first DNA componentsequence is, or has substantial sequence homology to, a chemicallyregulatable DNA sequence in a naturally occurring chemically regulatablegene from a plant and the second DNA component sequence is a codingsequence that is capable of being transcribed and translated to producea polypeptide resulting in a phenotypic trait. Preferably this secondDNA component sequence is adjacent to the first DNA coding sequence.Particularly preferred is such a chimeric DNA sequence wherein the firstDNA component sequence is, or has substantial sequence homology to, thechemically inducible DNA sequence in a PR protein gene, for example a PRprotein from a dicotyledonous plant, such as tobacco or cucumber.Examples thereof are mentioned above in Part D.

A second exemplified type of chimeric DNA sequence contains three DNAcomponent sequences, originating from two or more sources. In thesimplest embodiment this chimeric DNA sequence comprises the two-partDNA sequence as described above in Part E and a third DNA sequenceoriginating from at least one different source. More specifically, thistype of chimeric DNA sequence comprising a first DNA component sequencewhich is, or has substantial sequence homology to, a non-coding,chemically regulatable DNA sequence of a naturally occurring chemicallyregulatable gene, this first DNA component sequence being capable ofregulating the transcription of an associated DNA sequence in a plant orplant tissue, wherein this regulation is dependent upon a chemicalregulator, a second DNA component sequence which is, or has substantialsequence homology to, part but not all of a transcribable DNA sequencewith which the first component is associated in the naturally occurngchemically regulatable gene, and a third DNA component sequence capableof being transcribed in a plant or plant tissue. Preferably, thenaturally occurring chemically regulatable gene is a plant gene.

The second and third DNA component sequences will typically be codingsequences. The third DNA component may be derived from more than onenatural or synthetic source. The first two DNA components will typicallybe natural in origin. If the origin is a plant, it may be a monocot, adicot or a gymnosperm. In a preferred embodiment the second DNA sequencewill include the nucleotide sequence which codes for the signal peptideof the chemically regulatable gene in which the first two DNA sequencesoccur. If the chemically regulatable DNA sequence is associated withpart of the coding sequence of the gene from which it was derived, thethird DNA sequence must not only be in the proper orientation, but mustalso be in the proper reading frame with the second DNA sequence. Thisorientation can be achieved by techniques well known in the art.

Preferred are chimeric DNA sequences wherein the first and the secondDNA sequence components are those mentioned as preferred in Part Eabove. For example a preferred chimeric DNA sequence is one wherein thefirst DNA component sequence is, or has substantial sequence homologyto, the chemically inducible DNA sequence in a PR protein gene from adicotyledonous plant, for example from tobacco or cucumber. Examples ofPR protein genes arn mentioned above.

The chimeric genes described above embrace a variety of possibleconstructions. A chemically regulatable non-coding sequence can beassociated with a gene controlling flowering or fruit ripening; a geneeffecting tolerance or resistance to herbicides or to many types ofpests, for example fungi, viruses, bacteria, insects, nematodes, orarachnids; a gene controlling production of enzymes or secondarymetabolites; male or female sterility; dwarfness; flavor; nutritionalqualities; and the like. Using the present invention such traits can beenhanced by the farmer and gardener, which is no longer dependent onnatural factors alone. A phenotypic trait of particular interest forcontrol is the production of metabolites in tissue culture or abioreactor.

A preferred chimeric DNA sequence is a two or three component sequencewherein the coding DNA component sequence codes for a phenotypic trait,for example a trait selected from the group consisting of tolerance orresistance to a herbicide, fungus, virus, bacterium, insect, nematode orarachnid; production of secondary metabolites; male or female sterility;and production of an enzyme or other reporter compound. Particularlypreferred is a two or tree component chimeric DNA sequence wherein thecoding component sequence codes for tolerance or resistance toherbicides, for example codes for wild-tppe or herbicide resistantacetohydroxyacid synthase (AHAS), or wherein the coding componentsequence codes for resistance to insects, for example codes for Bacillusthuringiensis endotoxin (BT).

If the chimeric sequence is to be used as an assay for chemicalregulators, the phenotypic trait is preferably an assayable marker.Suitable markers include, but are not limited to, luciferase (LUX),chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase(NPT), nopaline synthase (NOS), octopine synthase (OCS),beta-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS), andBacillus thuringiensis endotoxin (BT). Preferred markers arebeta-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS), andBacillus thuringiensis endotoxin (BT).

A representative example of such a chimeric DNA sequence, described indetail in the examples, is a two-part chimeric DNA sequence whichcontains the 5′ flanking, non-coding sequence of the PR-1a gene. Whileone of the exemplified marker is the coding sequence for the GUS gene,any of the above mentioned markers could be used. The analogousthree-part chimeric sequence contains part of the coding sequence of thePR-1a gene. These constructions are particularly useful because theeffect of the chemical induction, i.e. beta-glucuronidase enzymeactivity, is easily detectable in plant cells or extracts thereof. Otherparticular embodiments, for example those which comprise the non-codingsequence of one of the tobacco β-1,3-glucanase genes and those whichcomprise the coding sequence for wild-type or herbicide resistantacetohydroxyacid synthase or for Bacillus thuringiensis endotoxin, aredescribed in Part O, Examples.

Preferred chimeric DNA sequence are two component or three componentchimeric DNA sequences wherein the first DNA component sequence is the5′ flanking region of the tobacco PR-1a gene and contains more thanabout 300, for example between 300 and 2000, preferably between 600 and1000, base pairs adjacent to the transcriptional start site.

Further preferred is a chimeric DNA sequence comprising three componentswherein the second DNA component sequence codes for a signal peptide,for example wherein the second DNA component sequence codes for apeptide which is, or has substantial sequence homology to, the signalpeptide from a PR protein gene, preferably the PR-1a gene.

The invention further embraces a method for the preparation of achemically regulatable chimeric DNA sequence comprising a firstnon-coding, chemically regulatable DNA component sequence which iscapable of regulating the transcription of an associated DNA sequence ina plant or plant tissue, wherein this regulation is dependent upon achemical regulator, and a second DNA component sequence capable of beingtranscribed in a plant or plant tissue, characterized in that the DNAcomponent sequences are ligated. Likewise, the invention embraces amethod for the prepartion of a chimeric DNA sequence comprising a firstDNA component sequence which is, or has substantial sequence homologyto, a noncoding, chemically regulatable DNA sequence of a naturallyoccurring chemically regulatable gene, this first DNA component sequencebeing capable of regulating the transcription of an associated DNAsequence in a plant or plant tissue, wherein this regulation isdependent upon a chemical regulator, a second DNA component sequencewhich is, or has substantial sequence homology to, part but not all of atranscribable DNA sequence with which the first component is associatedin the naturally occurring chemically regulatable gene, and a third DNAcomponent sequence capable of being transcribed in a plant or planttissue, characteized in that the DNA component sequences are ligatedconcurrently or consecutively.

G. VECTORS

Vectors, produced by standard techniques and comprising either thechemically regulatable DNA sequences described in part D or E or thechimeric DNA sequences described in Part F above, represent anadditional feature of the invention. Vectors are recombinant DNAsequences which may be used for isolation and multiplication purposes ofthe mentioned DNA sequence and for the transformation of suitable hostswith these sequences. Preferred vectors for isolation and multiplicationare plasmids which can be propagated in a suitable host microorganism,for example in E. coli. Preferred vectors for transformation are thoseuseful for transformation of plant cells or of Agrobacteria ForAgrobacterium-mediated transformation, the preferred vector is aTi-plasmid derived vector. For the direct DNA transfer into protoplasts,any of the mentioned vectors may be used. Appropriate vectors which canbe utilized as staring materials are known in the art. Suitable vectorsfor transforming plant tissue and protoplasts have been described bydeFramond, A. et al., Bio/Technology 1: 263 (1983); An, G. et al., EMBOJ. 4: 277 (1985); Potrykus, I. et al., supra; Rothstein, S. J. et al.,Gene 53: 153 (1987). In addition to these, many other vectors have beendescribed in the art which are suitable for use as starting materials inthe present invention.

The vectors which contain only the chemically regulatable DNA sequenceas described in Part D or E above can be used as intermediates for thepreparation of a vector containing the chimeric DNA sequence asdescribed in Part F above. The insertion of an appropriate sequence,which is capable of transcription, into such an intermediate vectorresults in a vector comprising a chimeric DNA sequence of the inventionthat can then be used to transform the desired plant tissue, protoplastor other host. Alternatively, a chimeric DNA sequence can be prepard andinserted into a suitable vector which is then used to transform thedesired plant tissue or other host.

The construction and multiplication of the vectors can be performed in asuitable host, for example, in E. coli. Suitable E. coli strains includeHB101, JM83, DH1, DH5, LE392 and the like. The vectors of the inventionmay be used as such in a direct gene transfer or a microinjectiontechnique. In certain instances it may be preferable to linearize thevector before use. Alternatively the vectors may be transferred to anAgrobacterium host. This transfer is accomplished by conventionaltechniques including biparental mating (Simon, R. et al., Bio/Technology1: 74 (1983)), triparental mating (Ditta, G. et al., Proc. Natl. Acad.Sci. USA 77: 7347 (1980)) or transformation (Holsters, M. et al., Mol.Gen. Genet. 163: 181 (1978)). Suitable strains of Agrobacterium includebut are not limited to A. tumefaciens LBA4404, CIB542 and C58Z707.

Preferred vectors are those comprising the preferred DNA sequencesmentioned in Parts D, E and F above. Furthermore a preferred vector isone that is functional in plant cells or in Agrobacterium or both.Particularly preferred are the vectors described in Part H, Examples.

H. PLANT TISSUES, PLANTS AND SEEDS

A further aspect of the invention are plant tissue, plants or seedscontaing the chimeric DNA sequences described above. Preferred are planttissues, plants or seeds containing those chimeric DNA sequences whichare mentioned as being preferred.

The cells of plant tissue are transformed with the vectors describedabove by any technique known in the art, including those described inthe references discussed above and by techniques described in detail inthe examples which follow. These techniques include direct infection orco-cultivation of plants or plant tissue with Agrobacterium. A verysuitable technique is the leaf disk transformation described by Horsch,R. B. et al., Science 225: 1229 (1985). Alternatively, the vector can betransferred directly, for example by electroporation, by microinjectionor by transformation of protoplasts in the presence of polyethyleneglycol (PEG), calcium chloride or in an electric field, as more fullydescribed above.

The cells transformed may originate from monocotyledenous ordicotyledonous plants and may contain one or more of the chemicallyregulatable chimeric genes of this invention. Thus, genes which, forexample, code for resistance or tolerance to herbicides and a variety ofinsect, viral, bacterial, fungal and other pests, for sterility, forsize, for flowering and fruit ripening, are introduced in the planttissue, and these cells or protoplasts ultimately regenerated intoplants in which these traits can be controlled by manipulations with achemical regulator. Alternatively cells can be propagated in tissueculture or in a bioreactor to produce enzymes or secondary matabolites.If an enzyme assay is desired, the coding section of the chimeric genemay, for example, comprise a LUX, CAT, NPT, NOS, OCS, GUS, AHAS or BTgene, as identified previously. Such chimeric genes containing achemically inducible sequence from a PR gene are a preferred embodimentof the invention because of the case of application of the regulator andthe ease of detection of the enzyme product.

Following transformation, the transformed cell or plant tissue isselected or screened by conventional techniques. The transformed cell orplant tissue contains the chimeric DNA sequence discussed above and isthen regenerated, if desired, by known procedures, including thosedescribed in the reference discussed above and in the examples whichfollow for both monocot and dicot plants. The species which can beregenerated by these techniques include, but are not limited to, maize,sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice,potato, eggplant and cucumber. The regenerated plants are screened fortransformation by standard methods. Progeny of the regenerated plants iscontinuously screened and selected for the continued presence of theintegrated DNA sequence in order to develop improved plant and seedlines. The DNA sequence can be moved into other genetic lines by avariety of techniques, including classical breeding, protoplast fusion,nuclear transfer and chromosome transfer.

I. ADVANTAGES AND USES

1. Chemical Regulation of Expression

The present invention offers a number of advantages and uses stemmingfrom the easily controlled regulatable expression in plants or planttissue of the chimeric genes contaning chemically regulatable DNAsequences. Regulation of genes acting by an anti-sense mechanism or ofgenes whose expression results in the production of a phenotypic traitis possible. Of particular importance are the ability to control thetime and rate of gene expression and the ease of effecting this control,either uniformly throughout the plant or in localized parts of theplant.

Effecting the control may be accomplished simply by applying thechemical regulator to the plant tissue, or to the plant or part of theplant in such a manner and in such an amount to regulate the chimericgene(s) whose expression in plant cells, plant tissues or plants isdesign For example, if the trait to be expressed is preferably expressedonly in the leaves, then spraying or dusting the leaves at a time whichoptimizes that expression in the leaves, and before any migration toother parts of the plant, may accomplish that objective easily andefficiently. Alternatively uniform expression throughout that part ofthe plant above ground may result from application to the entire plant(i.e., stem and both sides of the leaves). If expression in the roots isdesired, application to the seeds or the soil around the seeds or rootsis a possible method of regulation. Expression in a bioreactor isaccomplished quite easily, for example, by applying the chemicalregulator to the medium contacting the cells.

The ability to control the time, rate and/or gene expression of novelphenotypic traits in transgenic plants offers a number of usefuladvantages. For example, if tolerance by a detoxification mechanism to aherbicide or other pesticide is introduced into a plant, that trait canbe maximized by proper timing of the the application of the chemicalregulator. Thus, the regulator can be applied before, with or afterapplication of a herbicide or other pesticide, depending on which methodgives optimal tolerance. It is also possible now to regulate theproduction of compounds whose biosynthesis is controlled by endogenousor foreign genes. Upon chemical induction the production of suchproducts can be started at the desired time. The induced process couldbe a multi-step biosynthesis or a one-step conversion of a metabolite.

Another advantage of the present invention arises from the ability toregulate the developmental processes in plants at a desired time by theapplication of a regulating chemical. For example, the synchronizationof plant development (germination, tillering, sprouting, flowerformation, anthesis, fruit ripening, dry down, abscission etc.) can beaccomplished. In addition, normal plant development can be prevented.This can be accomplished by introducing, for example, a toxin gene whichwould interfere with the desired developmental stage, a DNA sequencewhich would block the developmental stage through an anti-sensemechanism, or a gene whose expression blocks the transition to a newdevelopmental stage and which can be chemically repressed to allowdevelopment to procced. One suitable utility is the induction of male orfemale sterility for the controlled hybridization of crops.

Additional advantages of the present invention are novel assayprocedures, preferably enzyme assay procedures. For example,identification of new chemical regulators can now be accomplished quiteeasily. Such assay methods involve the use of transformed plants orplant cells which contain a chimeric DNA sequence of this invention. Achemical is applied to the transformed host and the transcription of thetranscribable DNA sequence is measured against a control; transcriptionis usually detected in the form of a translation product or as an effectof such a product. The assay may be performed in a variety of ways, butis typically carried out using whole, regenerated plants (by applyingthe chemical to the plant) or to cultured cells or tissue (by applyingthe chemical to the cells or tissue) and subsequently supplying asubstrate for the enzyme activity. The product of enzyme activity isthen detected by standard methods, e.g., spectrophotometic measurement.

In particular the invention embraces a process for identifying achemical regulator which comprises transforming a host with a chimericDNA sequence as described above in Part C or with a vector containingsaid chimeric DNA sequence, applying a putative chemical regulator tothe transformed host, and measuring the expression of the phenotypictrait. A preferred process is such a process wherein the transformedhost is plant tissue or plant cells. Further preferred is a processwherein the chimeric DNA sequence is a sequence mentioned above as beingpreferred, for example wherein the first DNA component sequence of saidchimeric DNA is a chemically inducible sequence which is, or hassubstantial sequence homology to, a chemically inducible sequence of aPR protein gene, and wherein the phenotypic trait coded for by thechimeric DNA is an assayable enzyme marker.

Another feature of the invention is the development of novel methods toidentify other chemically regulatable DNA sequences. A vector containinga putative chemically regulatable DNA sequence is prepared, for example,from a host-selectable marker and a selectable or assayable marker.Suitable selectable markers include antibiotic resistance genes andherbicide resistance genes. Representative antibiotic resistance genesincludes those for hygromycin, kanamycin, chloramphenicol, bleomycin,puromycin, lincomycin, G418 and methotrexate. A particularly suitablegene for resistance to hygromycin is aminoglycoside phosphotransferaseIV. Suitable vectors for the transformation of plants containing thehygromycin resistance gene have been described by Rothstein, S. J. etal., Gene 53: 153 (1987). Examples of herbicide resistance genes encoderesistance to, for example, sulfonylureas, glyphosate, phosphinotricinand atrazine.

Suitable assayable markers include genes for enzymes, antigens,immunoglobulins and the like, as well as antibiotic resistance genes.Suitable enzyme markers include LUX, CAT, NPT, NOS, OCS, GUS, AHAS andBT. A particularly suitable enzyme is beta-1,3-glucuronidase (GUS)because of the ease of enzyme assay. The DNA sequence coding for theassayable marker can be inserted into the vector using conventionaltechniques.

The putative regulatable DNA sequence is typically inserted adjacent tothe assayable marker gene so that the expression of the assayable makeris under the control of the putative DNA sequence. The vector is thenused to transform plant tissue or another appropriate host. Transformedplant tissue or host is selected on the basis of the plant selectablemarker, typically antibiotic resistance. A chemical regulator is thenapplied to the plant tissue or other host following selection orfollowing regeneration of the transformed tissue. Induction orrepression of the assayable marker following the application of thechemical regulator identifies the putative DNA sequence as one which iscapable of regulating the transcription of an adjacent DNA sequencewherin the regulation is dependent on a chemical regulator. The assaycan be performed on whole, regenerated or transformed plants, forexample, by applying the chemical regulator to the leaves or other planttissue, and measuring the expression of the assayable marker.Alternatively the assay can be performed on transformed callus tissue orother host in cell culture, by applying the putative chemical regulatorto the callus or other culture and measuring the expression of theassayable marker.

In particular the invention embraces a process for identifiing achemically regulatable DNA sequence which comprises the steps of:

(a) transforming a host with a putative chemically regulatable DNAsequence or a vector containing said sequence, a second DNA sequencewhich is a host-selectable gene marker and a third DNA sequence whichcodes for a phenotypic trait;

(b) applying a chemical regulator to said transformed host; and

(c) measuring the expression or selecting for change in expression ofthe phenotypic trait coded for by the third DNA sequence.

Preferred is such a process wherein said third DNA sequence is ahost-selectable or an assayable gene marker, and a process wherein thesecond or third DNA sequence is a gene marker for herbicide resistanceor antibiotic resistance, for example selected from the group consistingof hygromycin, kanamycin, chloramphenicol, bleomycin, puromycin,lincomycin and methotrexate resistance genes, e.g. an aminoglycosidephosphotransferase IV hygromycin resistance gene.

Also preferred is such a process wherein the putative chemicallyregulatable DNA sequence is associated with, and preferably adjacent to,the third DNA sequence. Further preferred is a process wherein thetransformed host is plant tissue or plant cells.

Further preferred is such a process wherein the third DNA sequence codesfor an assayable enzyme marker selected from the group consisting ofluciferase (LUX), chloramphenicol acetyltransferase (CAT), neomycinphosphotransferase (NPT), nopaline synthase (NOS), octopine synthase(OCS), beta-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS),and Bacillus thuringiensis endotoxin (BT).

A further aspect of the invention are substantially pure chemicallyregulatable DNA sequence identified by the mentioned processes.

The present invention also provides a method for selecting transformedplant material. Plant material exposed to an exogenous DNA sequencecontaining a chemically regulatable DNA sequence for which a chemicalregulator is known and a second DNA sequence for a phenotypic trait istreated with the regulator and expression of the phenotypic traitsought. Observation of the trait confirms that transformation of theputative transformants has indeed occurred. Typical phenotypic traitsfor purposes of this method include the host selectable markers andassayable markers previously described.

In particular the invention embraces a method for selecting transformedplant cells or tissue which have been subjected to transforming DNAcomprising a first DNA component sequence that is chemically regulatableby a known chemical regulator and an associated second DNA sequencecoding for a phenotypic marker, which process comprises the steps of (a)treating putative transformants with said known chemical regulator and(b) selecting transformants according to expression of the phenotypictrait coded for by the second DNA sequence. A preferred method is such amethod wherein said phenotypic trait is antibiotic resistance, forexample resistance to hygromycin, kanomycin, chloroamphenicol,bleomycin, puromycin, lincoymcin, G418 and methotrexate, or an assayableenzyme marker, for example selected from the group consisting ofluciferase (LUX), chloramphenicol acetyltransferse (CAT), neomycinphosphotransferase (NPT), nopaline synthase (NOS), octopine synthase(OCS), beta-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS),and Bacillus thuringiensis endotoxin (BT).

2. Disease Resistance or Tolerance

With regard to the anti-pathogenic sequences described in part K below,the present invention provides a means for enhancing the ability of aplant, plant tissue or seed to withstand challenge by a pathogen. Thusthe present invention makes it possible to genetically engineer plantsfor enhanced resistance or tolerance to pathogens including, but notlimited to, viruses or viroids, e.g. tobacco or cucumber mosaic virus,ringspot virus or necrosis virus, pelargonium leaf curl virus, redclover mottle virus, tomato bushy stunt virus, and like viruses, fungi,e.g. Phythophthora parasitica or Peronospora tabacina, bacteria, e.g.Pseudomonas syringae or Pseudomonas tabaci, or aphids, e.g. Myzuspersicae.

3. Enhanced Exogenous Regulaton via Inactivation of EndogenousRegulation

The present invention provides a method for exogenous regulation of geneexpression in plants wherein the corresponding, native, endogenousregulation mechanism of the genes in the plant is renderednon-functional. In general, the method is applicable to any plantcapable, of being altered in a manner described herein, and isparticularly applicable to agronomically important plants such as maize,wheat, soybean, cotton, rapeseed, barley, rice, sorghum, sunflower,bean, beet and tobacco.

Certain genes in plants are regulated endogenously by at least onecorresponding signal transduction cascade (pathway), that is, theproduction in the plant cell of various regulating chemicals; e.g.,signal molecules. These molecules often are produced via a biosyntheticpathway in response to an external stimulus such as, for example, anecrotizing pathogen. In turn, these signal molecules regulate; i.e.,induce or repress, the expression of various genes in the plant. Forinstance, treatment of a plant such as tobacco by a necrogenic pathogen;e.g., TMV, or salicylic acid or 2-chloroethylphosphonic acid (Ethephon,Signa Chemicals, St. Louis, Mo.) initiates a process that leads to theaccumulation of high concentrations of salicylic acid (SA) in other,non-infected parts of the plant. SA is bound by receptors in or on thetarget cells. The signal is transduced intra-cellularly. SA thenactivates the coordinate induction of the expression of a set of atleast nine systemic acquired resistance (SAR) gene families, whichinclude the ten pathogenesis-related (PR) proteins of tobacco. Theexpression products of these gene families causes the plant target cellsto become resistant to attack by a side variety of agronomicallyimportant bacterial, fungal and viral pathogens. For example, transgenictobacco expressing high levels of PR-1a have reduced disease symptomsfollowing infection by oomycete fungi, including Peronospora tabacina(downy mildew) and Phytophora parasitica (black shank disease) (seeExamples 148-168).

Applicants have discovered that inactivating an endogenous signaltransduction cascade such that the expression of the target gene(s) iseffectively eliminated affords the exclusive exogenous control of thesegenes. For example, Applicants, having confirmed that SA is theendogenous signal molecule that mediates SAR in plants such asArabidopsis, tobacco and cucumber, have discovered that this signalcascade can be controlled; i.e., inactivated, disarmed or rendereddysfunctional, such that the induction of the target genes by SA isessentially eliminated. That is, the resultant concentration of thesignal molecule in the plant cell is insufficient to activate thepromoters of the signal-regulated genes. In turn, they have discoveredthat expression of the target genes can be induced by exogenousapplication of a chemical which acts downstream of the signaltransduction cascade, or otherwise acts independently of the SA pathway.

The signal cascade can be rendered non-functional in a number of ways.First, the plant cell can be stably transformed with a gene encoding anenzyme capable of metabolizing or inactivating the plant cell signal.The gene encoding such an enzyme may be derived from any organism; e.g.,microbe, plant or animal, or may be a truncated or synthetic gene,provided, however, that the gene is functional in plants. The gene canbe linked to a promoter functional in plants and which allows expressionat high levels in those cell types in which the subsequent exogenouschemical regulation is intended to be effected. In the alternative, apromoter may be used which drives expression at high levels in all ornearly all cell types. The promoter must be capable of functioningindependently of the signal; i.e., expression of the operably linkedgene(s) does not depend on the signal, and the exogenous chemical.Examples of suitable promoters include constitutive promoters such asthe CaMV 35S promoter, small subunit of RUBISCO, an enhanced 35Spromoter such as that described in Ray et al., Science 236: 1299-1302(1987), a double 35S promoter such as that cloned into pCGN2113 (ATCC40587), and any other constitutive promoter capable of functioning inthe plant tissue of interest.

In a preferred embodiment, a plant is transformed with nahG a gene whichencodes salicylate hydroxylase (SH). SH (E.C. 1.14.13.1) catalyzes theconversion of salicylate to catechol. Yamamoto et al., J. Biol. Chem.240(8): 3408-3413 (1965). This gene can be obtained from any soilmicrobe capable of growth on salicylate as sole carbon source. Examplesinclude Pseudomonas sp., e.g., ATCC 29351 and 29352, Pseudomonas cepaciaand Trichosporon cutaneum (Einarsdottir et al., Biochemistry 27:3277-3285 (1988)). A preferred source is Pseudomonas putida PpG7 TCC17485), wherein nahG on the 83 kilobase plasmid NAH7 is on of twooperons involved in the conversion of naphthalene to pyruvate andacetaldehyde (Yen et al., Proc. Natl. Acad. Sci. USA 79: 874-878(1982)). The 1305 base paid nucleotide sequence of the nahG codingregion and approximately 850 base pairs of the 3′ flanking sequence havebeen determined (You et al., Biochemistry 30: 1635-1641 (1991)).Approximately 200 base pairs of the 5′ flanking sequence also have beendetermined. See Schell, Proc. Natl. Acad. Sci. USA 83: 369-373 (1986).Methods of transforming plants are known, and are disclosed herein andin co-owned pending application Ser. No. 07/583,892, filed Sep. 14,1990, the relevant disclosure of which is incorporated herein byreference. Those skilled in the art could select an appropriatetransformation method depending upon the type of target plant.

Those skilled in the art will appreciate that other means can beemployed to achieve the same effect. For instance, a second methodinvolves the expression or overexpression in a transformed plant of agene encoding an enzyme which catalyzes the modification; e.g.,degradation, of a metabolic precursor of the signal molecule so that theplant is rendered incapable of producing the signal molecule. A thirdmethod involves the external application to the plant of antagonists ofthe target cell signal. Such antagonists compete with the cell signalfor the cell signal target site, but do not activate the responsegenerated by the cell signal. Instead, inhibition of the cell signalresponse is effected. In the case of salicylic acid, o-trimethylsilylbenzoic acid exhibits such an antagonistic effect when appliedexogenously to a plant. Further, aminoethoxyvinyl glycine andaminooxyacetic acid have been found to inhibit the ethylene cascade inplants, and that the ethylene response was restored upon subsequent,exogenous application of ethylene. See, Yang and Hoffman, Ann. Rev.Plant Physiol. 35:155-189 (1984).

Yet a fourth method involves the selection of plant mutants which failto respond to the selected cell signal, but which are responsive to thepredetermined exogenous chemical regulator. Methods of selecting mutantsfor a predetermined trait are known in the art. These include EMS,gamma-rays, T-DNA transposon insertion, and the like. A fifth methodinvolves the expression of antisense RNA to any gene involved in thesignal transduction cascade. This may include the expression ofantisense RNA to a gene involved in the biochemical pathway leading tothe synthesis of the cell signal, or in the alternative, to a geneencoding a receptor or other component of the pathway. See, Oeller etal., Science 254: 437-439 (1991). In either case, the cell signal whichis regulating particular genes or sets of genes is significantlyreduced. The cell signal also can be effectively rendered non-functionalby overexpressing sense transcripts of any gene involved in thetransduction cascade (pathway) utilizing a promoter functional in plantcells. Ths stratagem is based on the observation that the attemptedoverexpression of a gene in transgenic plants or plant cells can lead toa down-regulation of the homologous gene in the host plant as well asthe transgene. See, van der Krol et al., Plant Cell 2: 291-299 (1990).

In cases where two or more endogenous signal transduction cascades canregulate at least gene or gene family of interest, techniques todeactivate all cascades can be used, thereby rendering the genes ofinterest regulatable only by exogenous application of a chemicalregulator.

There are several signal transduction cascades known in plants.Representative examples include the phytohormones such as ethylene whichaffects fruit ripening and other responses (Guzman and Ecker, Plant Cell2: 513-523 (1990); light (Chong et al., Cell 58: 991-999 (1990); touch(Braam and Davis, Cell 60: 357 (1990); and gravity (Okada et al., Cell70: 369-372 (1992).

Once inactivation of the cell signal is achieved, the genes which arenatively regulated by the signal can be regulated exclusively by theexogenous application to the plant of a chemical regulator. In general,the chemical regulator, which can be naturally or non-naturallyoccurring in plants, functions “downstream” of the signal in thetransduction pathway, or functions completely independently; e.g., isnot involved in the pathway. In the case of the SA pathway, for example,representative chemical regulators capable of inducing expression of PRgenes include benzo-1,2,3-thiodiazole-7-carboxylate, n-propylbenzo-1,2,3-thiadiazole-7-carboxylate methylbenzol-1,2,3-thiodiazole-7-carboxylate, benzylbenzo-1,2,3-thiodiazole-7-carboxylate, andbenzo-1,2,3-thiodiazole-7-carboxylic acid N-sec-butylhydrazide. Thesecompounds are non-naturally occurring in plants.

Other chemicals encompassed by the present invention can be determinedby assaying the test chemical in the presence of a plant modified in amanner described above, which plant also contains an endogenous orheterologous reporter gene operably linked to a promoter regulatable bythe signal molecule. Since the modified plant is incapable of producingthe signal molecule in sufficient amounts to include expression of thereporter gene, no difference wlll be observed upon application to theplant with the chemical unless the chemical is capable of regulatingexpression of the reporter gene. Examples of reporter genes includeluciferase (LUX), chloramphenicol acetyltransferase (CAT), neomycinphosphotransferase (NPT), nopaline synthase (NOS), octopine synthase(OCS), beta-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS)and Bacillus thuringiensis endotoxin (Bt) (Williams et al.,Bio/Technology, 10: 540-543 (1992). The assay can be perfomed usingwhole plants or with plant tissue in an in vitro assay.

In certain situations, it would be desirable to regulate the expressionof various heterologous genes (transgenes) in transgenic plants. Forexample, the effectiveness of disease- or insect resistance intransgenic plants transformed with genes encoding disease- orinsect-resistant proteins, respectively, could be enhanced if the timingof the expression could be controlled See, e.g., Uknes, Plant Cell, 4:645-656 (1992); Ward et al., Plant Cell 3:1085-1094 (1991); Gould,Bioscience 38: 26-33 (1988); and Gould, TIBTECH 6: S15-S18 (1988). Also,the chemical regulation o developmental processes such as homeosis,germination, tillering, sprouting, flowering, anthesis, fruit ripening,and abscission offers several advantages such as the facilitatedproduction of hybrid seed, greater reduction of crop loss, and moregenerally, control of the growth and development of the plant by thefarmer. Thus, the present invention applies equally to transgenic plantscontaining heterologous genes, e.g., disease resistance genes includingPR and SAR genes, insect resistance genes such as Bt genes, and genesinvolved in developmental processes such as those described above. Italso includes genes encoding industrial or pharmaceutical biomaterialssuch as plastics and precursors thereof, perfumes, additives, enzymesand other proteins, and pharmaceutical, wherein the plant effectivelywould be used as a bioreactor, e.g., the two genes encoding productionof polyhydroxybutyrate, a thermoplastic (Poirer et al., Science 256:520-523 (1992). To practice this embodiment of the present invention,the heterologous gene of interest should be fused to a promoter capableof being regulated by the exogenous chemical regulator (eg. containing achemically regulatable DNA sequence) and for which activity, the signalis not required exclusively. In other words, the promoter can beregulatable by the signal, provided that it can be regulated by achemical regulator in the absence of a functional, endogenous signal.Examples include the PR-1a promoter such as those disclosed herein andin Williams et al., Bio/Technology 10: 540-543 (1992); Uknes et al., ThePlant Cell 5: 159-169 (1993); and Van de Rhee et al., Plant Cell 2:357-366 (1990), and other promoters isolated from chemically regulatedplant genes such as those described herein and in Payne et al., PlantMol. Biol. 11:89-94 (1988).

J. SIGNAL PEPTIDES

A signal peptide or a signal sequence is a special N-terminal amino acidsequence in a protein entering the endoplasmic reticulum. Such asequence in cukaryotic cells typically contains about 15 to 40 aminoacid residues, many of which are hydrophobic. The sequence is eventuallycleaved from the mature protein.

In the context of the present invention the signal peptide of achemically regulatable gene is useful in the construction of three-partchimeric constructions as described above. Such chimeric constructionscontain a first chemically regulatable sequence, a second DNA sequencecoding for a signal peptide in proteins, and a third sequence whichcodes for a phenotypic trait. Preferably the phenotypic trait is onethat is easily detected, for example in an assay procedure. Inclusion inthe chimeric construction of the DNA sequence which codes for a signalpeptide allows the product expressed by the third DNA sequence to bedirected away from the endoplasmic reticulum of the host cell to itsultimate target site.

Therefore, an additional feature of the invention is a signal peptidefrom the tobacco PR-1a protein, and a protein with an amino acidsequence having substantial sequence homology to this peptide. The aminoacid sequence for that peptide is as follows:

MetGlyPheValLeuPheSerGlnLeuProSerPheLeuLeuValSerThrLeuLeuLeuPheLeuValIleSerHisSerCysArgAla.

The present invention also embraces the DNA sequence which codes forthis peptide (see nucleotides 932-1021 of SEQ ID No. 1) and DNAsequences having substantial sequence homology to this sequence. Asnoted previously, the invention also embraces (1) DNA sequencescontaining the chemically regulatable sequence in combination with thesequence coding for the signal peptide and (2) three-part chimeric DNAsequences containing the chemically regulatable component, the codingsection for a signal peptide and a DNA sequence that is transcribed,preferably with translation.

K. ANTI-PATHOGENIC SEQUENCES

The present invention also embraces anti-pathogenic DNA sequences whichare capable of conferring enhanced disease resistance or diseasetolerance when expressed in a plant or plant tissue. This includescoding sequences for plant pathogenesis-related (PR) proteins asdescribed herein and sequences with substantial homology to these codingsequences.

Included within the scope of the present invention, in addition to thesequences exemplified specifically below and enumerated in the sequencelisting, are cDNA sequences which are equivalent to the enumeratedSequences which encode the given plant pathogenesis-related protein, andcDNA sequences which hybridize with the enumerated Sequences and encodea polypeptide having some degree of disease-resistant activity of thegiven plant pathogenesis-related protein (i.e. an anti-pathogenicsequence).

Equivalent cDNA sequences are those which encode the same protein eventhough they contain at least one different nucleotide from theenumerated sequence. As is well known in the art, the amino acidsequence of a protein is determined by the nucleotide sequence of theDNA. Because of the redundancy of the genetic code, i.e., more than onecoding nucleotide triplet (codon) can be used for most of the aminoacids used to make proteins, different nucleotide sequences can code fora particular amino acid. Thus, the genetic code can be depicted asfollows:

Amino Acid Codon Amino Acid Codon Phenylalanine (Phe) TTK Histidine(His) CAK Leucine (Leu) XTY Glutamine (Gln) CAJ Isoleucine (Ile) ATMAsparagine (Asn) AAK Methionine (Met) ATG Lysine (Lys) AAJ Valine (Val)GTL Aspartic acid (Asp) GAK Serine (Ser) QRS Glutamic acid (Glu) GAJProline (Pro) CCL Cysteine (Cys) TGK Threonine (Thy) ACL Trytophan (Trp)TGG Alanine (Ala) GCL Arginine (Arg) WGZ Tyrosine (Tyr) TAK Glycine(Gly) GGL Termination signal TAJ

Key: Each 3-letter deoxynucleotide triplet codon corresponds to atrinucleotide of mRNA, having a 5′-end on the left and a 3′-end on theright All DNA sequences given herein are those of the strand whosesequence corresponds to the mRNA sequence, with thymine substituted foruracil. The letters stand for the purine or pyrimidine bases forming thedeoxynucleotide sequence as follows:

A=adenine; G=guanine; C=cytosine; T=thymine

X=T or C if Y is A or G

X=C if Y is C or T

Y=A, G, C or T if X is C

Y=A or G if X is T

W=C or A if Z is A or G

W=C if Z is C or T

Z=A, G, C or T if W is C

Z=A or G if W is A

QR=TC if S is A, G, C or T;

QR=AG if S is T or C

J=A or G

K=T or C

L=A, T, C or G

M=A, C or T

The above shows that the amino acid sequence of the instant plantpathogenesis-related proteins can be prepared using different nucleotidesequences encoding the same amino acid sequence of the proteins.Accordingly, the scope of the present invention includes such“equivalent nucleotide sequences.”

cDNA sequences that hybridize with a given enumerated sequence andencode a polypeptide or protein having at least some degree of activityof the corresponding plant pathogenesis-related protein are those whichexhibit substantial sequence homology, as defined hereinabove, with theenumerated Sequence such that it hybridizes with the latter under lowstringency conditions. Such conditions are described in Examples 46 and51-58, below. Proteins translated from these hybridizable cDNA sequenceshave different primary structures from proteins translated from theenumerated Sequences. However, their respective secondary structures arethe same.

Also included as part of the present invention are plant tissues, plantsand seeds comprising anti-pathogenic sequences analogous to those planttissues, plants and seeds comprising chemically regulatable sequencesdescribed in part H above.

L. DIFFERENTIAL CLONING AND SCREENING TECHNOLOGY

A method has been conceived and developed which will allow efficientenrichment of sequences present in one population of molecules ingreater amounts than in another population. The method's greatestutility is in situations where the populations are very similar and thedifferentially present sequences represent a very small proportion ofthe population.

If two populations of clones are similar and one wishes to isolate thoseclones which are present in one population in higher amounts (i.e.“induced” or differentially regulated”), past techniques involvedscreening with probes from the two populations (+/− screening; St. Johnand Davis, Cell 16:443-452 (1979)), or enrichment of probes or mRNAs byhybridization and hydroxy-apatite (HAP) chromatography (Davis, et al.,Proc. Natl. Acad. Sci, USA 81: 2194-2198 (1984)). The first method has ademonstrated sensitivity limitation in that only clones present ingreater than about one in 2,000 will be detected. The second islaborious, technically difficult, and achieves enrichments of 20-50 foldat best.

The present method involves exploiting two recent developments inmolecular technology: the polymerase chain reaction (Saiki et al.,Science 239:487-491 (1988)) and biotin-avidin chromatography [Stahl, etal., Nuc. Acids. Res. 16: 3026-3038 (1988)). The polymerase chainreaction (PCR) allows simple synthesis of large amounts of DNA ofspecified sequence. Biotin-avidin chromatography allows the efficientseparation of molecules bearing a biotin affinity tag from thosemolecules which do not bear the tag.

In its general form, the technique consists of isolating single strandsof cDNA representing two different populations (“induced” vs“uninduced”), but of opposite cDNA polarity for the two populations,i.e. one of “sense” polarity relative to mRNA's, and the other itscomplement, or “anti-sense”, polarity relative to mnRNA's. The isolatedstrands from the “induced” population would have no affinity tag, whilethe strands of opposite polarity from the “uninduced” populations wouldhave stable affinity tags. When these two populations are hybridizedtogether, hybrids will form between complementary strands present in thetwo to populations. Those strands from the “induced” population whichhave no counterparts, or many fewer counterparts, in the “uninduced”population, remain single stranded.

Due to the presence of the affinity tag (in essence a handle) on thestrands of the “uninduced” population molecules, those strands and, mostimportantly, any hybrid molecules can be removed from the mixture byaffinity chromatography. This leaves only those “induced” moleculeswhich are not significantly represented in the “uninduced population.These “induced” molecules can then be cloned by standard means and serveas an enriched population from which to isolate “induced” clones;alternatively, the enriched molecules can be amplified individually andsequenced directly.

An alternate scheme is the same as described above except that itinvolves incorporating a labile affinity tag only on the “induced”population molecules, while the affinity tag on the “uninduced”population is stable. “Labile” in this case means that the affinity tagcan be removed at will, or be altered at will in such a way that it nolonger serves as an affinity tag. In this scheme all the molecules inthe hybridization mixture could bind to the affinity matrix, but onlythose “induced” molecules that are not hybridized to a complementary“uninduced” counterpart could be selectively recovered from the matrixfor subsequent cloning.

The advantage of the methods of the invention described above over thosepreviously described is the ability to isolate those genes which areturned on only to low levels, in specfic circumstances, and which mayplay a causative role in some important biological phenomenon.

The present invention teaches the cloning of SAR genes by differentialscreening of tissues induced and non-induced to the systemic acquiredresponse. SAR induction causes the transcription of genes in a proteinsynthesis-dependent fashion and also a protein synthesis-independentfashion. Two methods were used to clone specifically genes whose inducedtranscription is protein-synthesis independent. Firstly, cDNAs whichwere cloned by standard differential screening techniques were furtherscreened on SAR-induced RNA isolated with and without cycloheximide(CHX) pre-treatment. Secondly, a PCR-based “differential display”technique was used to identify SAR-induced, but protein synthesisindependent cDNAs directly. Differential display RNAs were prepared withand without SAR induction and CHX treatment. The use of CHX as aninhibitor of protein synthesis is well known in the art and is describedby Greenberg et al., Mol. Cell Biol. 6: 1050-1057 (1986), Lau andNathans, Proc. Natl. Acad. Sci. 84: 1182-1186 (1987), and Uknes et al.,Plant Cell 5: 159-169 (1993). Thus, a number of genes were cloned whichwere induced by the SAR response, yet expressed independently of proteinsynthesis. These cloned genes are likely signal transducers in thepathway leading from induction to the development of the resistantstate.

M. CHEMICAL REGULATORS

A chemical regulator is defined as a substance which regulatesexpression of a gene through a chemically regulatable DNA sequence undercertain circumstances as indicated in the definition of the term. Thesubstance, in ionic or neutral form, with or without solvating or othercomplexing molecules or anions, will usually be exogenous relative tothe system containing the chemically regulatable gene at the timeregulation is desired. The use of exogenous chemical regulators ispreferred because of the ease and convenience of controlling the amountof regulator in the system. However, the invention also includes the useof endogenous regulators, e.g., chemicals whose activities or levels inthe system are artificially controlled by other components in, or actingon, the system.

Chemical regulators include chemicals known to be inducers for PRproteins in plants, or close derivatives thereof. These include benzoicacid, salicylic acid, polyacrylic acid and substituted derivativesthereof; suitable substituents include lower alkyl, lower alkoxy, lowerallylthio and halogen. When applied to plant tissue, typically to theleaves of whole plants, increased levels of mRNA and PR proteins developin the plant tissue.

An additional group of regulators for the chemically regulatable DNAsequences and chimeric genes of this invention is based on thebenzo-1,2,3-thiadiazole structure and includes, but is not limited to,the following types of compounds: benzo-1,2,3-thiadiazolecarboxylicacid, benzo-1,2,3-thiadiazolethiocarboxylic acid,cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acidamide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, and derivativesthereof.

A preferred group of regulators includes, but is not limited to,benzo-1,2,3-thiadiazole-7-carboxylic acid,benzo-1,2,3-thiadiazole-7-thiocarboxylic acid,7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylicacid amide, benzo-1,2,3-thiadiazole-7-carboxylic acid hydrazide, andderivatives thereof.

Suitable derivatives encompass but are not limited to representatives ofsaid types of compounds wherein the benzo-1,2,3-thiadiazole moiety isunsubstituted or substituted by small substituents normally used inaromatic ring systems of agrochemicals such as lower alkyl, loweralkoxy, lower haloalkyl, lower haloalkoxy, lower alkylthio, cyano, nitroand halogen. Suitable derivatives further encompass, but are not limitedto, representatives of said benzo-1,2,3-thiadiazole compounds whereineither the carboxylic acid, the thiocarboxylic acid, the carboxylic acidamide or the carboxylic acid hydrazide functional group is unsubstitutedor substituted by aliphatic, araliphatic or aromatic residues. Suitableresidues encompass, but are not limited to, alkyl (especially loweralkyl), alkoxy (especially lower alkoxy), lower alkoxyalkyl,alkoxyalkoxyalkyl, cycloalkyl, cycloalkylalkyl, phenylalkyl (especiallybenzyl), naphthylalkyl, phenoxyalkyl, alkenyl, and alkinyl, wherein thealkyl part of the substituent is unsubstituted or substituted byhydroxy, halogen, cyano or nitro, and the aromatic part of thesubstituent is unsubstituted or substituted by small substituentsnormally used in aromatic ring systems in agrochemicals such as loweralkyl, lower alkoxy, lower haloalkyl, lower haloalkoxy, lower alkylthio,cyano, nitro and halogen.

Regulators based on the benzo-1,2,3-thiadiazole structure encompass allmolecular systems capable of releasing the molecule actually acting asthe regulator.

A preferred group of regulators based on the benzo-1,2,3-thiadiazolestructure includes benzo-1,2,3-thiadiazolecarboxylic acid, alkylbenzo-1,2,3-thiadiazolecarboxylate in which the alkyl group contains oneto six carbon atoms, and substituted derivatives of these compounds.Suitable substituents include lower alkyl, lower alkoxy, lower alkylthioand halogen. In particular, benzo-1,2,3-thiadiazole-7-carboxylic acidand its alkyl esters, e.g. methyl benzo-1,2,3-thiadiazole-7-carboxylate,are preferred inducers for the chimeric DNA sequences comprisingchemically regulatable DNA sequences isolated from PR protein genes. Thesyntheses of the mentioned chemical regulators and their utility asbiocides may be discerned from British Patent 1,176,799 and Kirby, P. etal., J. Chem. Soc. C 2250 (1970).

Derivatives of benzo-1,2,3-thiadiazole that may further be used asregulators according to the present invention are described in U.S.patent application Ser. No. 234,241 filed Aug. 18, 1988, which is herebyincorporated by reference.

Among the preferred species based on the benzo-1,2,3-thiadiazolestructure there may be mentioned, for example,benzo-1,2,3-thiadiazole-7-carboxylic acid, methylbenzo-1,2,3-thiadiazole-7-carboxylate, n-propylbenzo-1,2,3-thiadiazole-7-carboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide, and thelike.

An additional group of regulators for the chemically regulatable DNAsequences of this invention is based on the pyridine carboxylic acidstructure, such as the isonicotinic acid structure and preferably thehaloisonicotinic acid structure. Preferred are dichloroisonicotinicacids and derivatives thereof, for example the lower alkyl esters.Suitable regulators of this class of compounds are, for example,2,6-dichloroisonicotinic acid, and the lower alkyl esters theeof,especially the methyl ester.

A further aspect of the invention is therefore a process for regulatingtranscription of a chemically regulatable gene, which process comprisesapplying such a chemical regulator to plant tissue, plant or seedcontaining a chemically regulatable DNA sequence as described in Part A,B or C above. Preferred is such a process wherein the plant tissue,plant or seed contains a chemically regulatable DNA sequence mentionedabove as being preferred.

The chemical regulators may be applied in pure form, in solution orsuspension, as powders or dusts, or in other conventional formulationsused agriculturally or in bioreactor processes. Such formulations mayinclude solid or liquid carriers, that is, materials with which theregulator is combined to facilitate application to the plant, tissue,cell or tissue culture, or the like, or to improve storage, handling ortransport properties. Examples of suitable carriers include silicates,clays, carbon, sulfur, resins, alcohols, ketones, aromatic hydrocarbons,and the like. If formulated as a conventional wettable powder or aqueousemulsion, the regulator formulation may include one or more conventionalsurfactants, either ionic or non-ionic, such as wetting, emulsifying ordispersing agents.

The regulators may also be applied to plants in combination with anotheragent which is desired to afford some benefit to the plant, a benefitrelated or unrelated to the trait controlled by any chimeric gene whichis regulated by the regulator. For example, a regulator can be admixedwith a fertilizer and applied just before the expression of a transgenictrait unrelated to fertilization is desired. Or it can be combined witha herbicide and applied to mitigate the effect of the herbicide at thetime when such effect would otherwise be at a maximum.

As a liquid formulation the regulator may be applied as a spray to plantleaves, stems or branches, to seeds before planting or to the soil orother growing medium supporting the plant. Regulators can also be usedin bioreactor systems, regulation being achieved by a single addition ofregulator formulation to the reaction medium or by gradual addition overa predetermined period of time.

The regulator is applied in an amount and over a time sufficient toeffect the desired regulation. A preferred regulator is one which showsno, or only minimal phytotoxic or other deleterious effect on the plant,plant tissue or plant cells to which it is applied in the amountapplied.

Preferred DNA sequences among those described above in Part D or E andpreferred chimeric DNA sequences among those described above in Part Fare in particular those wherein the transcription is regulated by achemical regulator mentioned above, for example selected from the groupconsisting of benzoic acid, salicylic acid, acetylsalicylic acid,polyacrylic acid and substituted derivatives thereof, or selected fromthe group consisting of benzo-1,2,3-thiadiazolecarboxylic acid,benzo-1,2,3-thiadiazolethiocarboxylic acid,cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acidamide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, and derivativesthereof, or dichloroisonicotinic acid or a derivative thereof. Mostpreferred are those DNA sequences wherein the transcription is regulatedby the mentioned preferred chemical regulators, for example bybenzo-1,2,3-thiadiazole-7-carboxylic acid, methylbenzo-1,2,3-thiadiazole-7-carboxylate, n-propylbenzo-1,2,3-thiadiazole-7-carboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide,2,6-dichloroisonicotinic acid, or methyl 2,6-dichloroisonicotinate, inparticular methyl benzo-1,2,3-thiadiazole-7-carboxylate.

N. DEPOSITS WITH THE ATCC AND NRRC

The following deposits have been made with the American Type CultureCollection (ATCC), Rockville, Md.:

1) Plasmid pCGN783, ATCC number 67868, deposited Dec. 22, 1988.

2) Plasmid pCGN1540, ATCC number 40586, deposited Mar. 22, 1989.

3) Plasmid pCGN2113, ATCC number 40587, deposited Mar. 22, 1989.

4) Plasmiid pCIB/SAR8.2a, ATCC number 40584, deposited Mar. 22, 1989.

5) Plasmid pCIB/SAR8.2b, ATCC number 40585, deposited Mar. 22, 1989.

6) Plasmid pBScht28, ATCC number 40588, deposited Mar. 24, 1989.

7) Plasmid pBSGL117, ATCC number 40691, deposited Oct. 19, 1989.

8) Plasmid pBSPER1, ATCC number 40686, deposited Oct. 19, 1989.

9) Plasmid pBSGL134, ATCC number 40690, deposited Oct. 19, 1989.

10) Plasmid pBSGL167, ATCC number 40834, deposited Jul. 3, 1990.

11) Phage lambda tobcDNAGL153, ATCC number 40694, deposited Oct. 19,1989.

12) Phage lambda tobcDNAGL161, ATCC number 40695, deposited Oct. 19,1989.

13) Plasmid pBSPERB24, ATCC number 40687, deposited Oct. 19, 1989.

14) Plasmid pBSPERB25, ATCC number 40688, deposited Oct. 19, 1989.

15) Plasmid pBSCL2, ATCC number 40835, deposited Jul. 3, 1990.

16) Plasmid pBSTCL226, ATCC number 40838, deposited Jul. 3, 1990.

17) Plasmid pBSPR-4a, ATCC number 75016, deposited Jun. 5, 1991.

18) Plasmid pBSPR-4b, ATCC number 75015, deposited Jun. 5, 1991.

19) Plasmid pAGL2, ATCC number 75048, deposited Jul. 12, 1991.

20) Plasmid pAPR1C-1, ATCC number 75049, deposited Jul. 12, 1991.

21) Plasmid pSLP1, ATCC number 75047, deposited Jul. 12, 1991.

22) Plasmid pATL12a, ATCC number 75050, deposited Jul. 12, 1991.

23) Plasmid pBSGL125, ATCC number 40692, deposited Oct. 19, 1989.

24) Plasmid pBSGL148, ATCC number 40689, deposited Oct. 19, 1989.

25) Plasmid pBSGL135, ATCC number 40685, deposited Oct. 19, 1989.

26) Phage lambda tobcDNAGL162, ATCC number 40693, deposited Oct. 19,1989.

27) Plasmid pBSPER25, ATCC number 40692, deposited Oct. 19, 1989.

28) E. coli HB101/pTJS75-Km, ATCC number 67628, deposited Feb. 11, 1988.

29) Plasmid pBS-PR1013Cla, ATCC number 40426, deposited on Feb. 11,1988.

30) Plasmid pBS-PR1019, ATCC number 40427, deposited on Feb. 11, 1988.

31) Zea mays suspension culture 6-2717-GC, ATCC number 40326, depositedon May 20, 1987.

32) Plasmid pBS-Gluc39.1, ATCC number 40526, deposited on Dec. 29, 1988.

33) Plasmid pBS-Gluc39.3, ATCC number 40527, deposited on Dec. 29, 1988.

34) Plasmid pBScucchi/chitinase, ATCC number 40528, deposited on Dec.29, 1988.

35) Plasmid pBSGL6e, ATCC number 40535, deposited on Jan. 18, 1989.

36) Plasmid pCIB2001/BamChit, ATCC number 40940, deposited on Dec. 20,1990.

37) Plasmid pBScucchrcht5, ATCC number 40941, deposited on Dec. 20,1990.

The following deposits have been made with the Agricultural ResearchCulture Collection, International Depositing Authority, 1815 N.University Street, Peoria, Ill. 61604:

38) Plasmid pWCI-1, NRRL number NRRL-B21097, deposited on May 24, 1993.

39) Plasmid pWCI-2, NRRL number NRRL-B21098, deposited on May 24, 1993.

40) Plasmid pWCI-3, NRRL number NRRL-B21099, deposited on May 24, 1993.

41) Plasmid pWCI-4, NRRL number NRRL-B21100, deposited on May 24, 1993.

42) Plasmid pAtPR1-P, NRRL number NRRL B-21169, deposited Jan. 5, 1994.

The deposits have been made in accordance with the Budapest Treaty. Inaddition, Applicants declare that all restrictions on the availabilityof the deposits to the public will be irrevocably removed upon thegranting of a U.S. patent.

O. SUMMARY OF EXPERIMENTAL PROTOCOLS FOR CHEMICALLY REGULATABLESEQUENCES

The chimeric genes of the present invention contain chemicallyregulatable DNA sequences from any source, natural or synthetic, incombination with one or more transcribable DNA sequences; usually atleast part of the transcribable sequence will be translated into aphenotypic trait, although the invention also embraces chimeric geneswhich operate through an anti-sense mechanism. While the followingdescription and many of the examples are directed to chemicallyregulatable DNA sequences found naturally in a plant genome, thisinvention applies equally to such sequences which occur in other naturalsystems, for example, bacterial, viral and animal systems, sincetechniques are well known to isolate such sequences from their naturalsystems. Furthermore, sequences derived from synthetic or othernon-natural systems are similarly embraced.

A chemically regulatable DNA sequence is isolated from the source inwhich it exists naturally or synthetically and, if necessary,characterized by conventional methods. If the DNA sequence is isolatedfrom a naturally occurring gene, this gene is activated by anappropriate regulator, either by a chemical or other known regulator forthe gene. RNA resulting from that activation is isolated and is used toconstruct a cDNA library. This library is used for differentialscreening using radiolabelled cDNA generated both from (1) RNA isolatedfrom the activated system and (2) RNA isolated from a second system notactivated by the regulator. This differential screening is a particularaspect of the present invention, as mentioned above. The clonescontaining the chemically regulated cDNA are then isolated and arepreferably sequenced at this point. The cDNA clones are then used toidentify and isolate a genomic clone from a genomic library whichgenomic clone comprises the chemically regulatable DNA sequence. Thisgene is preferably sequenced and the chemically regulatable DNA sequenceis functionally mapped by subcloning fragments of this gene, ligatingthem to a reporter gene and evaluating their activity in transgenicplant material or in transient plant expression systems.

For the PR protein genes of tobacco a lambda genomic library isconstructed using standard techniques, and clones comprising chemicallyregulatable DNA sequences are identified and purified. One of theseclones is characterized. Restriction fragments carrying the chemicallyregulatable DNA sequence are identified; for the PR-1a gene these genefragments include a ClaI fragment of about 20,000 base pairs, a 6500base pair HindIII fragment, and a 3600 base pair EcoRI fragment. TheHindIII fragment contains about 500 coding base pairs and about 6000non-coding bases in the 5′ flanking region adjacent to the transitionalstart site. Some of these fragments are subcloned into plasmids for useas sources of DNA for further subcloning and full characterization,i.e., restriction mapping, DNA sequencing and identification of thetranscriptional start site of the gene. For the PR-1a gene the 3600 basepair EcoRI fragment is directly subcloned from a lambda genomic cloneand subsequently subcloned to a final clone of about 1200 base pairsflanked by XhoI and PstI sites. This clone contains a chemicallyinducible DNA sequence from the PR-1a gene and a portion of the adjacentcoding region for that gene; this portion includes the coding sequencefor the signal peptide of the PR-1a protein gene.

Likewise genomic clones are isolated which code for the basic form ofbeta-1,3-glucanase. Two clones named 39.1 and 39.3 are characterized,and restriction fragments comprising chemically regulatable DNAsequences identified.

Using the vectors of this invention, these clones can then be used forthe preparation of chimric genes containing thee parts as discussedpreviously. These chimeric DNA sequences contain the chemicallyregulatable sequence, part of the transcribable DNA and a third DNAsequence from a foreign source. Alternatively the clones can be furthermanipulated, e.g., by site directed mutagenesis, to remove all or mostof any coding fragment from the parent gene prior to attachment to acoding sequence from a foreign source to prepare a two-part chimericgene as described above. In a preferred embodiment that part of thechimeric gene which is not the chemically regulatable sequenceconstitutes a reporter gene for an easily observed or detectedphenotypic trait The following examples illustrate the genes forbeta-1,3-glucuronidase, wild-type and herbicide resistantacetohydroxyacid synthase, and Bacillus thuringiensis endotoxin, but avariety of other reporter genes can be envisioned as described above. Ina further preferred embodiment the coding component DNA sequence of thechimeric gene codes for tolerance or resistance to herbicides or forresistance to insects. This embodiment is exemplified by the mentionedgenes for acetohydroxyacid synthase and for Bacillus thuringiensisendotoxin.

The chimeric genes and vectors containing these genes can be introducedinto plant cells by a variety of techniques which give rise totransformed cells, tissue and plants or to cell cultures useful inbioreactors. Several techniques are described in detail in the exampleswhich follow, directed to both monocotyledons and dicotyledons. Some ofthese methods, included here for enabling purposes, are the subject ofother patent applications, in particular the U.S. patent applicationSer. No. 056,506, filed May 29, 1987, and the U.S. patent applicationSer. No. 165,665, filed Mar. 8, 1988 (incorporated by reference hereinin their entirety).

The transformed plant material can then be treated with a chemicalregulator and the expression of the phenotypic reporter gene observedand/or measured This kind of system provides an easy screening device toidentify the regulatory activity of particular chemicals. The inventionalso concerns an improved assay of beta-1,3-glucuronidase (GUS)enzymatic activity which allows the screening of a large number ofsamples with high precision and in a short period of time. In particularthis assay comprises reaction of plant tissue extracts in samples ofless than 0.5 ml containing 1.5 to 3 mM 4-methyl umbelliferylglucuronide at around 37° C. for 1 to 5 hours and determining theconcentration of the fluorescent indicator released.

The quantitative determination of steady-state levels of RNA is anessential step in the analysis of gene expression. To that end a primerextension assay has been developed. This method of the inventioninvolves labeling a gene specific oligonucleotide to high specificradioactivity, hybridizing the oligonucleotide to an RNA sample andextending the hybrid with reverse transcriptase. The extension productsare separated on denaturing acrylamide gels and visualized byautoradiography. The benefits of this method over previous assaysinclude ease of probe preparation, simplicity of the assay protocol andthe time required to carry out the assay. This primer extension assay isas sensitive and quantitative as S1 mapping.

The primer extension assay as applied under the particular reactionconditions described below in the examples is optimized for thedetermination of the level of PR-1 mRNA in total RNA extracted from TMVinfected tobacco leaves. The PR-1 mRNA is expressed as 1% of the mRNA inthese leaves based on quantitative analysis of the primer extension dataand the frequency of cDNA clones in a cDNA library derived from thisRNA. The improvements of the method of the invention relative to thosepreviously described comprise the labelling of the oligonucleotide to ahigh specific activity, the decreased molar amount of probe used in theassay (typically about 0.01 to 0.05 pmol), optimized hybridization andelongation time, and optimized nucleotide triphopsphate concentrations.

The invention therefore embraces a method for the determination of theamount of a specific RNA in a solution of RNA comprising

(a) labeling a RNA specific primer oligonucleotide of a length ofbetween 12 and 18 nucleotides to high specific radioactivity,

(b) hybridizing the RNA with the labeled oligonucleotide at aconcentration of between 0.1 and 20 nM for between 2 minutes and 24hours at a temperature around 37° C.,

(c) elongating the primer oligonucleotide with reverse transcriptase inthe presence of nucleotide triphosphates at a concentration of between0.003 and 1 mM for between 1 and 120 minutes, and

(d) separating and visualizing the elongation products withautoradiography.

EXAMPLES A. INTRODUCTION

The following examples further illustrate the present invention. Theyare in no way to be construed as a limitation in scope and meaning ofthe claims.

Enzyme reactions are conducted in accordance with the manufacturer'srecommended procedures unless otherwise indicated. The chimeric genesand vectors of the present invention are constructed using techniqueswell known in the art. Suitable techniques have been described inManiatis, T. et al., “Molecular Coning”, Cold Spring Harbor Laboratory,New York (1982); Methods in Enzymology, Volumes 68, 100, 101 and 118(1979, 1983, 1983 and 1986, respectively); and “DNA Cloning”, Glover, D.M. Ed. IRL Press, Oxford (1985). Medium compositions have been describedin Miller, J. H., “Experiments in Molecular Genetics”, Cold SpringHarbor Laboratory, New York (1972), as well as the references previouslyidentified.

1. Media Used in the Examples

SH-0 medium: Medium of Schenk, R. U. et al., Can. J. Bot., 50: 199-204(1972); without hormones. SH medium can be liquid or solidified with0.8% agar or with 0.5% GelRite®. The medium is normally sterilized byheat in an autoclave at 230-250° F. for 15-20 mins.

SH-30 medium: SH-0 medium containing 30 m Dicamba.

SH-45 medium: SH-0 medium containing 45 m Dicamba.

RY-2 medium: Medium of Yamada, Y. et al., Plant Cell Reports, 5: 85-88(1986).

OMS medium: Medium of Murashige, T. and Skoog, F., Physiologia Plantarum9: 473 (1968). The medium can be solidified with 0.8% agar or agarose orwith 0.5% GelRite®.

TABLE I Macroelements^(a), microelements^(a) and Fe-EDTA are as given inthe literature: KM medium according to Kao, K.N. et al., Planta 126:105-110 (1965); N6 medium according to Chu et al., Scientia Sinica 18:659 (1975). Composition of Media Used KM-8p^(b,c,d) N6 Organics andvitamins^(e) [mg/l]: biotin 0.01 pyridoxine-HC1 1.00 0.5 thiamine-hcl10.00 0.1 nicotinamide 1.00 nicotinic acid 0.10 0.5 folic acid 0.40D-Ca-pantothenate 1.00 P-aminobenzoic acid 0.02 choline chloride 1.00riboflavin 0.20 Vitamin B 12 0.02 glycine 0.10 2.0 Sugars and sugaralcohols [g/l]: sucrose 0.25 30.0 glucose 68.40 mannitol 0.25 sorbitol0.25 cellobiose 0.25 fructose 0.25 mannose 0.25 rhamnose 0.25 ribose0.25 xylose 0.25 myo-inositol 0.10 Final pH 5.8 5.6 Sterilization filterautoclaved ^(a) Macroelements are usually made up as a 10 X concentratedstock solution, and microelements as a 1000 X concentrated stocksolution. ^(b) Citric, fumaric and malic acid (each 40 mg/l finalconcentration) and sodium pyruvate (20 mg/l final concentration) areprepared as a 100 X concentrated stock solution, adjusted to pH 6.5 withNH₄OH, and added to this medium. ^(c) Adenine (0.1 mg/l finalconcentration), and guanine, thymidine, uracil, hypoxanthine andcytosine (each 0.03 mg/l final concentration) are prepared as a 1000 Xconcentrated stock solution, adjusted to pH 6.5 with NH₄OH, and added tothis medium. ^(d) The following amino acids are added to this mediumusing a 10 X stock solution (pH 6.5 with NH₄OH) to yield the given finalconcentrations: glutamine (5.6 mg/l), alanine, glutamic acid (each 0.6mg/l), cysteine (0.2 mg/l), asparagine, aspartic acid, cystine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine (each 0.1mg/l). ^(e) Vitamin stock solution is normally prepared 100 Xconcentrated.

2. Materials Used in the Examples

Agarose: Preparation and purification of agarose are described byGuiseley and Renn, “The Agarose Monograph”, Marine Colloids Division FMCCorp., 1975. Agarose is one of the constituents of agar. Commerciallyavailable agar normally consists of a mixture of neutral agarose andionic agaropectin with a large number of side groups. Usually a certainnumber of of side chains remains intact and determines thephysicochemical properties of the agarose such as gel formation andmelting temperature. Low-melting agarose, especially SeaPlaque® agarose,is a preferred solidifying agent.

Casein hydrolysate: Casein Hydrolysate—Enzymatic Hydrolysate from bovinemilk, Type 1, Sigma Co., PO. Box 14508, St. Louis, Mo. 63178, USA.

Cellulase RS: Cellulase RS, Yakult Honsha Co. Ltd., 1.1.19Higashi-Shinbashi, Minato-ku, Tokyo, 105 Japan.

GelRite®: GelRite Gellan Gum, Scott Laboratories Inc., Fiskerville, R.I.02823, USA.

GeneScreen Plus®: Cat. No. NEF 976, NEN Research Products, 549 AlbanySt., Boston, Mass. 02118, USA.

IBI Random primer kit: ‘Prime Time’ random primer kit, IntemationalBiotechnologies Inc., PO. Box 1565, New Haven, Conn. 07606, USA.

Nalgene® filter: Nalge Co., Division of Sybron Corp. Rochester, N.Y.14602, USA.

Pectolyase Y-23®: Seishin Pharmaceutical Co. Ltd., 4-13 Koami-cho,Nihonbashi, Tokyo, Japan.

Parafilm®: Parafilm® laboratory film—American Can Co. Greenwich, Conn.06830, USA.

SSC: 1.54 mM NaCl, 0.154 mM sodium citrate.

Spin column: Sephadex® G25 prepacked column, Cat.No. 100402, BoehringerMannheim Biochemicals, Piscataway, N.J., USA.

TAE buffer and TBE buffer: Tris-acetate buffer and Tris-borate buffer,respectively—common buffers for electrophoresis, see Maniatis et al.,supra.

B. GENERAL TECHNIQUES

This group of examples describes general manipulations used to carry outthe following detailed examples.

Example 1 Ligation in Agarose

Following restriction digestion of plasmid DNA and electrophoreticseparation of the fragments on a low melting TAE gel, the bandscontaining appropriate fragments are precisely excised and heated to 65°C. to melt the agarose. 2-5 l are added to 15 l water and the solutionis left at 65° C. for 10 minutes. This solution is cooled to 37° C. andleft for five minutes to equilibrate to temperature. 2 l of 10×ligasebuffer (200 mM Tris, pH 8.0, 100 mM MgCl₂, 100 mM DTT, 10 mM ATP) areadded along with 1 l T4 DNA ligase (New England BioLabs), and thissolution is allowed to solidify and incubate at 15° C. overnight.

Example 2 Transformation From Agarose

The agarose containing the appropriate DNA is melted by incubating at65° C. for 10 minutes. 10 l of this solution are added to 30 l of TEbuffer (10 mM Tris pH 7.5, 1 mM EDTA), mixed and allowed to stand atroom temperature. Frozen competent cells (E. coli strain DH5) are placedon wet ice to thaw. The diluted DNA solution is added to 200 l of cellsand allowed to stand on ice for 20 minutes. The cells containing the DNAare then heat-shocked for 90 seconds at 41° C. The cells are then leftat room temperature for 10 minutes. 0.8 ml of SOC medium (Hanahan, D.,J. Mol. Biol. 166: 557-580 (1983)) is added and the culture is incubatedat 37° C. for one hour. 100 l of the culture is plated on LB plates(Miller, supra) containing 100 g/ml ampicillin (L-amp) and the platesare incubated overnight at 37° C. Positive colonies are picked andrestreaked to a second L-amp plate and the plates are incubatedovernight at 37° C.

Example 3 Labelling DNA Restriction Fragments

DNA is treated with the appropriate restriction enzymes and fragmentsare separated by electrophoresis on a low-gelling temperature agarosegel. A band containing the fragment of interest is excised and the DNApurified by standard techniques. 50 ng of the DNA fragment is labelledysing the IBI Random primer kit “Prime time” according to themanufacturers directions.

Example 4 Southern Blotting

3 g of tobacco DNA is digested with various restriction enzymes underthe conditions suggested by the supplier. The DNA is extracted withphenol, precipitated with ethanol and then resuspended in gel loadingbuffer (15% ficoll, 0.025% bromophenol blue, 10 mM EDTA, pH 8). Samplesare loaded and electrophoresed on a 0.5% agarose gel at 5 V/cm until thebromophenol blue dye reaches the end of the gel. The DNA is transferredto Gene-Screen Plus (DuPont) using the alkaline transfer procedure asdescribed by the supplier. Pre-hybridization, hybridization and washingare according to the manufacturer's recommendation. Hybndization isdetected by autoradiography.

Example 5 Molecular Adaptors

A typical molecular adaptor is the sequence

5′-GGGATCCCrGCA-3′ (SEQ ID No. 47)

for the conversion of a PstI site to a BamHI site. This molecule issynthesized on an Applied Biosystems Synthesizer usingB-cyanoethylphosphoramidite chemistry and purified by reverse-phaseHPLC. About 2 g of this oligonucleotide is kinased according to Maniatiset al., supra p. 125. The oligonucleotide solution is heated to 65° C.in a water bath and allowed to cool to room temperature over a period ofabout 30 minutes. An approximately 10-fold molar excess of this annealedadapter is added to the digested DNA along with 10×ligase buffer, T4 DNAligase, and an appropriate amount of water. A typical reaction is:

DNA to be adapted: 1-2 l (˜1 pmol)

Adapter: 1 l (˜10 pmol)

10×ligase buffer: 1 l

T4 DNA ligase: 1 l

Water: 5-6 l

This solution is incubated at 12-15° C. for 30 minutes, and heated to65° C. for 30 minutes to inactivate the ligase. The salt concentrationand volume are adjusted for the appropriate restriction digest and theadapted DNA is digested to expose the adapted “sticky end.”Unincorporated adapters are removed either by electrophoresis on anagarose gel or by sequential isopropanol precipitations.

Example 6 Primer Extension Mapping A. Synthesis and 5′ End Labeling ofPrimers for Primer Extension

The following primer oligomers are synthesized using an AppliedBiosystems Synthesizer and β-cyanoethylphosphoramidite chemistry:

PR-1: 5′-ATAGTCTTGTTGAGAGTT-3′ (SEQ ID No. 48)

GUS: 5′-TCACGGGTTGGGGTTTCTAC-3′ (SEQ ID No. 49)

AHAS: 5′-AGGAGATGGTTTGGTGGA-3′ (SEQ ID No. 50)

BT: 5′-ATACGTCTACTATCATAGT-3′ (SEQ ID No.51)

The oligonucleotides are purified by reverse-phase high pressure liquidchomatography (HPLC). 5 pmol of each oligo is kinased (Maniatis, T. etal., supra, at p. 125) using 200° C. of ³²P-ATP (6000 Ci/mmol, 10 Ci/l).After incubation at 37° C. for 30 minutes, the reacton is diluted to 100l, extracted with phenol/chloroform and then precipitated three timeswith 50 g carrier RNA. The final precipitate, is resuspended in1×reverse-transcriptase buffer (50 mM Tris-HCl, pH 7.5, 40 mM KCl, 3 mMMgCl₂) at a concentration of 2 nM. The specific activity of the labeledoligonucleotide is determined to be about 3×10⁶ Cvcpm/pmol.

B. Total RNA Preparation

Total RNA is prepared essentially as described by Lagrimini, L. M. etal., Proc. Natl. Acad. Sci. USA 84: 7542 (1987). Tissue is ground underliquid nitrogen in a mortar and pestle. The ground tissue is added togrinding buffer (Lagrimini et al., supra) using 2.5 ml per gram tissue.An equal volume of phenol is then added and the emulsion is homogenizedin a Brinkman polytron. A one-half volume of chloroform is added and theemulsion is gently mixed for 15 minutes. The phases are separated bycentifugation and the aqueous phase is removed RNA is precipitated bythe addition of sodium acetate to 0.3 M and 2.5 volumes ethanol. Theprecipitate is collected by centrifugation and resuspended in 2 mlsterile water. Lithium chloride is added to a final concentration of 3 Mand left at 4° C. overnight. The precipitate is collected bycentrifugation and the pellet is washed with ice-cold 80% ethanol. Thepellet is dried and resuspended in 500 l sterile water. Theconcentration of this total RNA preparation is determinedspectrophotometrically.

Alternatively, RNA is extracted from callus as described above exceptthat the callus tissue is cut into cubes approximately 3 mm in size, andadded to pre-chilled mortars and pestles for grinding in liquid nitrogenprior to the polytron step.

C. Primer Extension

50 g of total RNA is lyophilized in a 500 l Eppendorf tube. The RNA isresuspended in 30 l of radiolabeled probe solution and heated to 70° C.for 10 minutes. The tube is slowly cooled to 37° C. and allowed toincubate overnight. Without removing the tube from the 37° C. waterbath, 2 l of 10×reverse-transcriptase buffer (500 mM Tris-HCl, pH 7.5,400 mM KCl, 30 mM MgCl₂), 1 l 5 mg/ml bovine serum albumin, 5 l 100 mMdithiothreitol, 5 l 10×dNTPs (10 mM of each dNTP in H₂O), 3 l H₂O, 2 lRNasin (80 units), and 2 l reverse transcriptase (400 units) are addedand the reaction is incubated at 37° C. for 30 minutes. To stop thereaction, 5 l of 3 M sodium acetate, pH 5, and 150 l absolute ethanolare added. The tube is left at −20° C. for 30 minutes, the precipitateis collected by centrifugation, washed with 80% ethanol and allowed toair-dry. The precipitate is resuspended in 10-20 l of loading dye (90%formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 1 mM EDTA) andthe extension products are separated on a 6% sequencing gel (Maniatis,T. et al., suyra). Extension products are visualized by autoradiography.

Example 7 S1 Nuclease Mapping

The plasmid pBS-PR1013Cla is digested with SfaNI, dephosphorylated withcalf intestinal phosphatase and kinased with ³²P-ATP. Following phenolextraction and ethanol precipitation, the DNA is digested with BstEIIand the 300 bp fragment from 750 to 1035 of FIG. 1 is isolated afterelectrophoresis on a low gelling temperature agarose gel. The probe isresuspended in formamide hybridization buffer (Berk, A. J. et al., Cell12, 721 (1977)) at a concentration of about 2 nM. The specific activityof the probe is about 5×10 Cvcpm/pmol.

Lyophilized, total RNA (50 g) is dissolved in 30 l of the probesolution, and the tubes are heated to 65° C. for 10 minutes, thenallowed to hybridize overnight at 48° C. S1 nuclease treatment and gelelectrophoresis are essentially as described, using an S1 concentrationof 400 units/ml and an incubation temperature of 30° C. The appropriateS1 nuclease concentration is determined in pilot experiments.

Example 8 Mapping the Transcriptional Start Site

The transcriptional start site for the PR-1a gene is determined by acombination of S1 nuclease mapping and primer extension analysis. Anautoradiogram of a primer extension experiment using either RNA isolatedfrom TMV-infected leaves or an mpl9 subclone of the XhoI-PstI fragmentas a template and a 17 base oligonucleotide complementary to positions1025 to 1042 of the PR-1a sequence as a specific primer is examined. Theprimer itself is labeled at the 5′ phosphate, therefore the size of theextension product will be identical to the size of the correspondingband in the sequencing lanes. The appearance of two strong bands iscorresponding to positions 902 and 903 and a weak band at position 901of the genomic clone suggests transcription initiating at either ofthese positions. However, primer extension analysis alone cannot be usedto identify the 5′ end of a mRNA. For instance, the mRNA may contain a5′ end that has been spliced from an upstaeam location.

To determine conclusively the 5′ end, high resolution S1 nucleasemapping is used in conjunction with primer extension. An SfaNI fragmentis labeled at the 5′ end and digested with BstEII to yield a strandspecific probe extending from position 759 to 1040. This probe is usedto map the 5′ end of PR-1a transcripts in RNA isolated from TMV-infectedtobacco leaves. A major band of 137±2 bases is found which correspondsto positions 901 to 905 of the genomic clone. In high resolution S1experiments, where the digestion products are electrophoresed along witha size standard of sequencing reactions performed on the probe, threebands are visualized corresponding to positions 901, 902 and 903. Theseresults confirm the primer extension analysis and place the 5′ end ofthe PR-1 mRNA at either position 901, 902 or 903. With regard totranscription initiation, one possible interpretation of these resultsis that RNA polymerase begins transcription at either base 901, 902 or903 with more or less equal probability. However, since eukaryotictranscription favors initiation at an A, a more likely explanation forthe apparent multiple 5′ ends is that the PR-1a mRNA begins at position903 (an A) and the PR-1b and -1c mRNAs begin each at one of the otherpositions on their corresponding genes.

C. PROTEIN IDENTIFICATION AND CHARACTERIZATION

The PR proteins relevant to these examples are isolated, purified andsequenced, for the first time in some cases and in accordance withliterature procedures in other, for the purpose of allowing theisolation of the corresponding cDNA's and ultimately for confirming theidentities of their cDNA's and chemically inducible genes.

Example 9 General Techniques for Peptide Generation, Purification, andAutomated Sequencing A. Reduction and Alkylation

Purified, lyophilized protein is dissolved in 6 M guanidine-HClcontaining 1 M Tris-HCl, pH 8.6, 10 mM EDTA. Dithiothreitol is added to20 mM and 4-vinylpyridine is added to a final concentration of 50 mM.The sample is then incubated for 1.5 hours under nitrogen. Thepyridylethylated material is desalted on HPLC using an Aquapore phenylcolumn (2.1×10 cm, Brownlee). The column is eluted with a linear, 5-80%gradient of acetonitrile/isopropanol (1:1) in 0.1% trifluoroacetic acid(TFA).

B Cyanogen Bromide Cleavage and Removal of Pyroglutamate

Cyanogen bromide cleavage is performed in situ according to Simpson, R.J. et al., Biochem. Intl. 8: 787 (1984). Digestion of PR-1 protein withpyroglutamate aminopeptidase (Boehringer Mannheim) is carried outaccording to Allen, G., Plant Sci. Lett. 26: 173 (1982).

C. LysC Digestion

Protein is digested with endoproteinase Lys-C (Boehringer Mannheim) in0.1 M Tris-HCl, pH 8.5, for 24 hours at room temperature using anenzyme:substrate ratio of 1:10. Resulting peptides are isolated by HPLCusing an Aquapore C-8 column (1×22 cm, Brownlee) eluted with a linearacetonitrile/isopropanol (1:1 ratio) gradient (0 to 60%) in 0.1% TFA.

D. Trypsin Digestion

Digestion with trypsin (Cooper) is performed in 0.1 M ammoniumbicarbonate, pH 8.2, containing 0.1 M calcium chloride for five hours at37° C. using an enzyme:substrate ratio of 1:100. Peptides generated areseparated on HPLC using the same conditions as with the Lys-C peptidesor performed in 0.1 M Tris-HCl pH 8.5 for 24 hours at 37° C. using anenzyme to substrate ratio of 1:50. Peptides are isolated by HPLC using aVydac C-18 column (2.1×150 mm) with a linear 0 to 60%acetonitrile:isopropanol (1:1) gradient in 0.1% TFA.

E. Sequencing

Automated Edman degradations are performed with an Applied Biosystems470A gas-phase sequencer. Phenylthiohydantoin (PTH) amino acids areidentified using an Applied Biosystems 120A PTH analyzer.

Example 10 Purification of PR-1a and PR-1b Protein

Plants of Nicotiana tabaccum cv. Xanthi are grown in a glasshouse andinfected when eight weeks old by gently rubbing the leaves with asuspension of a common strain (UI) of TMV (0.5 g/ml). Leaves areharvested seven days after infection and the intracellular fluid (ICF)is washed out of the leaves and collected according to Parent, J. G. etal., Can. J. Bot. 62: 564 (1984). 250 ml of ICF are concentrated to 50ml by lyophilization and loaded on an Ultragel ACA54 column equilibratedwith Tris-HCl, pH 8.0, and 1 mM EDTA. Eluates are analyzed byelectrophoresis on 10% polyacrylamide gels. Fractions containing PR-1proteins are pooled, lyophilized, resuspended in 3 ml water and thendialyzed overnight against water. This preparation is further purifiedby HPLC anion exchange chromatography on a TSK-DEAE 5PN column. Thecolumn is eluted with a 0-0.6 M NaCl gradient in 50 mM Tris-HCl, pH 8.0,1 mM EDTA. Fractions are analyzed by polyacrylamide gel electrophoresis(PAGE). PR-1b elutes first from the column at 0.18 M NaCl and PR-1aelutes at 0.28 M NaCl. Fractions containing each protein are pooled,dialyzed against water and lyophilized. The purity of the PR-1a andPR-1b protein preparation is confirmed by reverse-phase HPLC using anAquapore phenyl column (2.1×100 mm, Brownlee) and eluting with a linearacetonitrile/isopropanol (1:1) gradient (5-80%) in 0.1% TFA.

Example 11 Protein Sequence Determination

Purified, lyophilized PR-1a protein is dissolved in 6 M guanidine-HClcontaining 1 M Tris-HCl, pH 8.6, 10 mM EDTA. Dithiothreitol is added to20 mM and 4-vinylpyridine is added to a final concentration of 50 mM.The sample is then incubated for 1.5 hours under nitrogen. Thepyridylethylated material is desalted on HPLC using an Aquapore phenylcolumn (2.1×10 cm, Brownlee). The column is eluted with a linear, 5-80%gradient of acetonitrile/isopropanol (1:1) in 0.1% trifluoroacetic acid(TFA).

Automated Edman degradations are performed with an Applied Biosystems470A gas-phase sequencer. Phenylthiohydantoin (PTH) amino acids areidentified by an Applied Biosystems 120A PTH analyzer.

Cyanogen bromide cleavage is performed in situ according to Simpson, R.J. et al., Biochem. Intl. 8: 787 (1984). Digestion of PR-1 withpyroglutamate aminopeptidase (Boehringer Mannheim) is carried outaccording to Allen, G., Plant Sci. Lett. 26: 173 (1982).

PR-1a is digested with endoproteinase Lys-C (Boehringer Mannheim) in 0.1M Tris-HCl, pH 8.5, for 24 hours at room temperature using an enzyme tosubstrate ratio of 1:10. Peptides are isolated by HPLC using an AquaporeC-8 column (1×22 cm, Brownlee) by a linear acetonitrile/isopropanol (1:1ratio) gradient (0 to 60%) in 0.1% TFA.

Digestion of the N-terminal Lys-C peptide with trypsin (Cooper) isperformed in 0.1 M ammonium bicarbonate, pH 8.2, containing 0.1 Mcalcium chloride for five hours at 37° C. using an enzyme to substrateratio of 1:100. The two peptides generated are separated on HPLC usingthe same conditions as with the Lys-C peptides.

Example 12 Purification and Sequence of PR-1a and PR-1b Protein

A correlation of the DNA sequence of three cDNA clones with the PR-1a,-1b and -1c proteins is originally made by Cornelissen, B. J. C. et al.,supra based on a comparison of the published protein sequence data ofthree peptides derived from PR-1a (Lucas, J. et al., EMBO J. 4: 2745(1985)) and the primary structure of the protein deduced from the cDNAclones. However, the cDNA clone designated as PR-1a is truncated at the5′ end and can be compared to only two of the three peptides with amismatch of one residue. The encoded amino acid sequence deduced fromcDNA as prepared and analyzed in the example below, and the PR-1a cDNAsequence from Pfitzner, U. M. et al., supra, mismatch the publishedprotein sequence at the tryptophan residue as reported. They also do notmatch at three other positions of the amino-terminal protein sequence.This anomaly places the previous identification of the PR-1 clones inquestion.

In order to confirm the identity of our cDNA clones as either PR-1a,PR-1b or PR-1c, a large portion of the primary structrre of the purifiedPR-1a and PR-1b protein and peptides derived from the proteins isdetermined by amino acid sequencing. This data is then compared to theprotein sequence deduced from the nucleotide sequence of the cDNA's inorder to identify which of the cDNA clones corresponds to which protein.Plants of Nicotiana tabacum cv. Xanthi are grown in a glasshouse andwhen eight weeks old are infected by gently rubbing the leaves with asuspension of a common strain (UI) of TMV (0.5 g/ml). Leaves areharvested seven days after infection and the intracellular fluid (ICF)is washed out of the leaves and collected according to Parent, J. G. etal., Can. J. Bot. 62: 564 (1984). 250 ml of ICF are concentrated to 50ml by lyophilization and loaded on an Ultragel ACA54 column equilibratedwith Tris-HCl, pH 8.0, and 1 mM EDTA. Eluates of this column areanalyzed by electrophoresis on 10% native polyacrylamide gels. Fractionscontaining PR-1 proteins are pooled, lyophilized, resuspended in 3 mlwater and then dialyzed overnight against water. This preparation isfurther purified by HPLC anion exchange chromatography on a TSK-DEAE 5PNcolumn. The column is eluted with a 0-0.6 M NaCl gradient in 50 mMTris-HCl, pH 8.0, 1 mM EDTA. Fractions are analyzed by polyacrylamidegel electrophoresis (PAGE). PR-1b elutes first from the column at 0.18 MNaCl and PR-1a elutes at 0.28 M NaCl. Fractions containing each proteinare pooled, dialyzed against water and lyophihzed. The purity of thePR-1a and PR-1b protein preparation is confirmed by reverse-phase HPLCusing an Aquapore phenyl column (2.1×100 mm, Brownlee) and eluting witha linear acetonitrile/isopropanol (1:1) gradient (5-80%) in 0.1% TFA.

Protein sequence is derived from either the deblocked amino terminus ofthe intact protein, from peptides derived from cyanogen bromide cleavageof the proteins, from peptides derived from LysC digestion of theproteins or from peptides derived from trypsin digestion of the proteinusing techniques detailed above or using other methods known in the art.Using standard techniques, the amino acid sequence of several peptidesderived from either PR-1a or PR-1b was determined.

Example 13 Purification and Sequence of PR-R Major and PR-R Minor

Plants of Nicotiana tabacum cv. Xanthi are grown in a glasshouse andinfected when eight weeks old by gently rubbing the leaves with asuspension of a common strain (U1) of TMV (0.5 g/ml). Leaves areharvested seven days after infection and the intracellular fluid (ICF)is washed out of the leaves and collected according to Parent, J. G. etal., Can. J. Bot. 62: 564 (1984). 250 ml of ICF are concentrated to 50ml by lyophilization and loaded on an Ultragel ACA54 column equilibratedwith Tris-HCl, pH 8.0, and 1 mM EDTA. Eluates are analyzed byelectrophoresis on 10% polyacrylanide gels. Fractions containing thePR-R protein and several minor contaminating proteins are pooled,lyophilized, resuspended in 3 ml water and then dialyzed overnightagainst water. This preparation is further purified by HPLC reversephase chromatography using a Brownlee Aquapore phenyl column (2.1×100mm). The column is eluted with a linear gradient of 20 to 80%acetonitrile:isopropanol (1:1) in 0.1% trifluoroacetic acid using a flowrate of 50 l/minute over 80 minutes. It is found that the major proteinspresent are isoforms of PR-R which are given the names PR-R major, forthe most abundant protein eluting at 46 minutes and PR-R minor for theless abundant protein eluting at 47.5 minutes. The peaks containing PR-Rmajor and PR-R minor are collected and the samples are reduced todryness. The proteins are then resuspended in 6M guanidine-HCl, 1MTris-HCl, reduced and alkylated as described above and subjected toautomated sequencing as described above.

A summary of the data obtained is presented below.

PR-R Major: ATFDIVNKCTYTWAAASPGGGRR (SEQ ID No. 52)

PR-R Minor: ATFDIVNQCTYTVWAAASPGGGRQLN (SEQ ID No. 53)

Example 14 Purifcation and Sequence of PR-P and PR-Q

Plants of Nicotiana tabacum cv. Xanthi are grown in a glasshouse andinfected when eight weeks old by gently rubbing the leaves with asuspension of a common strain (UI) of TMV (0.5 g/ml). leaves areharvested seven days after infection and the intracellular fluid (ICF)is washed out of the leaves and collected according to Parent, J. G. etal., Can. J. Bot. 62: 564 (1984). 250 ml of ICF are concentrated to 50ml by lyophilization and loaded on an Ultragel ACA54 column equilibratedwith Tris-HCl, pH 8.0, and 1 mM EDTA. Eluates are analyzed byelectrophoresis on 10% polyacrylamide gels. Fractions from the UltragelACA54 chromatography containing PR-O, PR-P, PR-Q and PR-R are pooled andconcentrated by lyophilization. The proteins are further purified bypolyacrylamide gel electrophoresis followed by electroblotting onto PVDFmembrane (Matsudaira, P., 1987, J. Biol. Chem. 261: 10035-10038). Theblotted protein bands containing PR-P and PR-Q are excised and treatedwith 0.5% PVP-40 in 100 mM acetic acid according to the AppliedBiosystems User Bulletin No. 36, Mar. 21, 1988. Deblocking of theprotein is carried out with pyroglutamate aminopeptidase as describedand the protein is sequenced from the PVDF by automated Edmandegradation as described above.

The sequences of the amino terminus of the PR-P and PR-Q proteins aredescribed below.

To obtain protein sequence from peptides derived from PR-P and PR-Q, thefractions from the Ultragel column, which contain PR-proteins, arepooled, lyophilized, dissolved in water and then dialyzed against waterprior to chromatography on DEAE-Sephacel. The DEAE-Sephacel column isequilibrated with 50 mM Tris-HCl (pH 8.0), 1 mM EDTA and eluted with alinear, 0 to 0.4 M gradient of sodium chloride. Fraction 6, whichcontains a mixture of PR-R major, PR-R minor, PR-P, PR-Q, PR-O andseveral minor contaminants is lyophilized and then resuspended indistilled water.

The proteins from Fraction 6 are further purified by HPLC using areverse phase phenyl column and eluted with a linear 20-80% gradient ofacetonitrile:isopropyl alcohol (1:1) in 0.1% trifluoroacetic acid (TFA).PR-P and PR-Q proteins co-eluted as a single peak which is collected andconcentrated almost to dryness in vacuo resuspended in 10 mM ammoniumacetate, pH 7.0 and applied to a Brownlee Labs AX-300 HPLC ion-exchangecolumn (2.1 mm×10 cm) equilibrated in 10 mM ammonium acetate (pH7.0).The proteins are eluted from this column using a linear gradient of 10to 250 mM ammonium acetate (pH 7.0). PR-P and PR-Q elute as singledistinct peaks at ca. 75 mM and ca. 95 mM ammonium acetate,respectively.

The protein is resuspended in 6M guanidine-HCl, 1M Tris-HCl and reducedand alkylated as described above. Peptides are generated by trypsindigestion, separated and sequenced as described above.

Example 16 Purification and Protein Sequence of PR-2, PR-N and PR-O.

Plants of Nicotiana tabacum cv. Xanthi.nc are grown in a glasshouse andinfected when eight weeks old by gently rubbing the leaves with asuspension of of a common strain (U1) of TMV (0.5 g/ml). Leaves areharvested seven days after infection and the intercellular fluid (ICF)is washed out of the leaves and collected according to Parent, J. G. etal., Can. J. Bot. 62: 564 (1984). 250 ml of ICF are concentrated to 50ml by lyophilization and loaded on an Ultragel ACA54 column equilibratedwith Tris-HCl, pH 8.0 and 1 mM EDTA. Eluates are analyzed byelectrophoresis on 10% polyacrylamide gels. Fractions containingPR-proteins as determined by gel electrophoresis are pooled,lyophilized, dissolved in water and dialyzed against water prior tochromatography on DEAE-Sephacel. The DEAE-Sephacel column isequilibrated with 50 mnM Tris-HCl (pH 8.0), 1 mM EDTA and eluted with alinear 0 to 0.4 M gradient of sodium chloride. Fraction 6, whichcontains a mixture of PR-Rmajor, PR-Rminor, PR-P, PR-Q, PR-O and severalminor contaminants, is lyophilized. Fraction 3, which contains a mixtureof PR-2, PR-N and several minor contaminants, is also collectedseparately and concentrated by lyophilization.

PR-O is further purified from other Fraction 6 proteins by firstresuspending Fraction 6 in 2 mls water and then separating the proteinsby HPLC reverse phase chromatography using a Vydac phenyl column(4.6×250 mm). Proteins are eluted using a linear 20-80% gradient ofacetonitrile:isopropanol (1:1) in 0.1% triflouroacetic acid. The resultsof this purification step reveal that the mixture contained at leastnine proteins. One of these proteins, eluting in one run at 51 minutesis PR-O as determined by gel electrophoresis. The peak containing PR-Ois collected and concentrated by lyophilization. The protein isresuspended in 6 M guanidine-HCl, 1M Tris-HCl and reduced and alkylatedas described above. Peptides are generated by trypsin and LysC digestionand are purified as described above. Protein sequence is determined asdescribed in Example 9, above. A summary of the sequencing data isprovided below.

The proteins PR-2 and PR-N are purified from Fraction 3 using a BrownleeAquapore AX-300 (2.1×100 mm) anion exchange column. The proteins areeluted from the column using a linear gradient of 10 mM to 350 mMammonium acetate pH 7.0. PR-N eluted at 37.5 minutes and PR-2 eluted at50.0 minutes as single, uniform peaks. The identity of the proteins isconfirmed by gel electrophoresis. The proteins are collected,concentrated by lyophilization and then reduced and alkylated asdescribed above. Peptides are generated by trypsin digestion, purifiedand sequenced as described in Example 9 above.

Example 17 Purification and Sequencing of PR-4

Plants of Nicotiana tabacum cv. Xanthi.nc are grown in a glasshouse andinfected when eight weeks old by gently rubbing the leaves with asuspension of a common strain (U1) of TMV (0.5 g/ml). Leaves areharvested seven days after infection and the intercellular fluid (ICF)is washed out of the leaves and collected according to Parent et al.,Can. J. Bot., 62: 564 (1984). 250 ml of ICF is concentrated to 50 ml bylyophilization and loaded on an Ultragel ACA54 column equilibrated withTris-HCl, pH 8.0 and 1 mM EDTA. Eluates are analyzed by electrophoresison 10% polyacrylamide gels. Fractions containing PR-proteins asdetermined by gel electrophoresis are pooled, lyophilized, dissolved inwater and dialyzed against water prior to chromatography onDEAE-Sephacel. The DEAE-Sephacel column is equilibrated with 50 mMTris-HCl (pH 8.0), 1 mM EDTA, and eluted with a linear 0 to 0.4 Mgradient of sodium chloride. Fraction 5, which contains PR-4 and severalminor contaminants, is lyophilized.

PR-4 is further purified from other Fraction 5 proteins by firstresuspending Fraction 5 in 2 mls water and then separating the proteinsby HPLC reverse phase chromatography using a Vydac phenyl column(4.6×250 mm). Proteins are eluted using a linear 20-80% gradient ofacetonitrile:isopropanol (1:1) in 0.1% trifluoroacetic acid. A peakfluting at approximately 24 minutes is determined by gel electrophoresisto contain PR-4 protein. This peak is collected and lyophilized andresuspended in 0.1 M Tris-HCl, pH 8.0. The protein is digested withtrypsin for 48 hours at 30° C., then incubated with 3 M guanidine-HCl, 1M Tris-HCl, and 20 mM dithiothreitol for 30 minutes at room temperature.(The protein is not reduced and pyridylethylated prior to digestion.)Tryptic peptides are purified and sequenced as described above.

Alternatively, PR-4 is purified by directly fractionating ICF on theVydac phenyl reverse phase column as described above. PR-4 co-eluteswith PR-1a, b, and c. This fraction is concentrated almost to drynessunder vacuum, and resuspended in 10 mM ammonium acetate (pH 7.0). PR-4is separated from PR-1 isoforms on a Brownlee Labs AX-300 HPLC ionexchange column (2.1 mm×10 cm), equilibrated with 10 mM ammonium acetate(pH 7.0). PR-4 is not retained on the column, while PR-1a, b, and c didbind. The purified PR-O is incubated in 6 M guanidine-HCl, 1 M Tris-HI(pH 8.6), 10 mM EDTA, 20 mM dithiothreitol for 1 hour at 37° C. 4-vinylpyridine is added and the incubation continued at room temperature for 1hour. The modified protein is desalted on a Brownlee labs PH-300 columnwith a linear gradient of 0-80% acetonitrile:isopropanol in 0.1%trifluoroacetic acid. Digestion with endoprotease Asp-N is carried outin 0/1 M Tris-HCl pH 8.0 for 4.5 hours at room temperature. Peptides arepurified and sequenced as described above.

Example 18 Purification and Protein Sequence of CucumberChitinase/Lysozyme

A pathogen-inducible chitinase protein is isolated from infectedcucumber leaves as described (Metraux, J. P. et al., Physiological andMolecular Plant Pathology, 33: 1-9 (1988)). Peptides are generated fromthis homogeneous protein preparation and sequenced essentially asdescribed above.

Example 19 Purification and Protein Sequence of the Cucumber Peroxidase

The pathogen-induced, acidic, cucumber peroxidase protein is purified asdescribed by Smith, J. A., Ph. D. Thesis, Department of Botany and PlantPathology, Michigan State University, Lansing, Mich. (1988), from theuninfected leaves of cucumber plants that had been infected seven dayspreviously with a spore suspension of Colletotrichum lagenarium. Thepurified protein is reduced and alkylated and amino acid sequence fromthe amino terminus and from peptides derived from either LysC or trypsindigestion is determined as described in Example 9 above.

D. PRODUCTION OF CLONES RELATED TO CHEMICALLY REGULATED SEQUENCES

This group of examples describes clones prepared as a result of geneisolation and identification of the chemically regulatable sequences andchimeric genes containing those sequences.

Example 20 Preparation of tobchrPR1013

Nuclei are isolated from leaves of Nicotiana tabaccum by first freezing35 grams of the tissue in liquid nitrogen and grinding to a fine powderwith a mortar and pestle. The powder is added to 250 ml of grindingbuffer (0.3 M sucrose, 50 mM Tris, pH 8, 5 mM magnesium chloride, 5 mMsodium bisulfite, 0.5% NP40) and stirred on ice for 10 minutes. Thismixture is filtered through six layers of cheesecloth, and the liquid istransferred to centrifuge bottles and subjected to centrifugation at700×g for 10 minutes. The pellets are resuspended in grinding buffer andrecentrifuged. The pellets are again resuspended in grinding buffer andrecentrifuged. The pellets are again resuspended in grinding buffer andthis suspension is layered over a 20 ml cold sucrose cushion containing10 mM Tris, pH 8, 1.5 mM magnesium chloride, 140 mM sodium chloride, 24%sucrose, 1% NP40. These tubes are centrifuged at 17,000×g for 10minutes. The pellets at this stage contain mostly nuclei and starchgranules. High molecular weight DNA is isolated from the nucleiessentially according to Maniatis, T. et al., supra. The DNA ispartially digested with MboI and ligated into the BamHI site of theEMBL3 cloning vector. Approximately 300,000 plaques are screened with alabeled PR-1b cDNA probe. Fifty positive clones are isolated andcharacterized. Positive plaques are purified and characterized byrestriction analysis. These clones are classified into eight distinctgroups by restriction mapping. One of the clones is identified astobchrPR1013. A partial restriction map of tobchrPR1013 is shown in FIG.1. Isolation of DNA and DNA manipulations are essentially as describedby Maniatis, T. et al., supra.

Example 21 Preparation of pBS-PR1013Cla

A ClaI fragment from the clone tobchrPR1013 is subcloned into theBluescript plasmid (Stratagene Cloning Systems), resulting inpBS-PR1013Cla (see FIG. 2). The 2 kb XhoI-BglII fragment frompBS-PR1013Cla is sequenced and positions 913 to 1711 (see SEQ ID No. 1)are found to match perfectly the sequence of a cDNA clone clone forPR-1a isolated by Pfitzner, U. M. et al., supra. Possible rearrangementsof the tobacco DNA during the cloning procedure are ruled out byanalyzing predicted restriction fragments from genomic DNA. Based onsequence information and a restriction map of pBS-PR1013Cla, digestionof genomic tobacco DNA with either EcoRI, HindIII, XhoI+BglII, orHindIII+PstI should generate fragments of 3.2 kb, 6.7 kb, 2.0 kb or 6.4kb, respectively, which contain the PR-1a gene. To test this prediction,3 g of tobacco chromosomal DNA is digested with the appropriate enzymesand electrophoresed on a 0.7% agarose gel. The DNA is transferred to anylon membrane and probed with a labeled XhoI-BstEII restrictionfragment from the PR-1a gene. As a control, pBS-PR1013Cla DNA isdigested and electrophoresed on the same gel. As predicted, the EcoRI,HindIII, XhoI+BglII, and HindIII+PstI digests produce bands of theexpected molecular weight which comigrate with the control bands.Therefore, the DNA contained in the tobchrPR1013 clone represents acontiguous piece of DNA in the tobacco genome.

Example 22 Preparation of pBS-PR1013Eco

A. The plasmid pBS-PR1013Eco is constructed by subcloning the 3.6 kbEcoRI fragment containing the PR-1a gene from tobchrPR1013 (Example 11)into the unique EcoRI site of the bluescript plasmid. Bluescript plasmid(catalog no. 21203) is obtained from Stratagene Cloning Systems, LaJolla, Calif. The structure of pBS-PR1013Eco is confirmed by bothrestriction mapping and DNA sequencing. This construction of the plasmidis shown in FIG. 3.

B. Alternatively, the plasmid pBS-PR1013Cla is digested with EcoRI andthe 3.6 kb fragment containing the PR-1a gene is isolated. ThepBluescript is digested with EcoRI, mixed with and ligated to thepreviously isolated 3.6 kb EcoRI fragment. This construction of theplasmid is shown in FIG. 4.

Example 23 Preparation of pBS-PR1013Eco Pst

The plasmid pBS-PR1013Eco is digested with PstI and the DNA fragmentsseparated on a 2.5% low-gelling temperature agarose gel. The largefragment containing the bluescript vector and a 3.0 kb fragment of thetobacco DNA are precisely excised and heated to 65° C. to melt theagarose. One of these plasmids is selected as the plasmid pBS-PR1013EcoPst. The structure of this subclone is verified by sequence analysis.The construction of this plasmid is shown in FIG. 5.

Example 24 Preparation of pBS-PR1013Eco Pst Xho

A preliminary restriction map of pBS-PR1013Eco is established. An XhoIrestriction site is located ˜1200 bp upstream from the PstI site. Theplasmid pBS-PR1013Eco Pst is digested with XhoI and the fragments areseparated on 0.9% low-gelling temperature agarose gel. Two fragments arevisualized with sizes of 3.9 kb and 1.7 kb. The band containing the 3.9kb fragment is carefully excised from the gel and ligated in agaroseessentially as described above. The agarose is melted and the DNAtransformed into competent E. coli strain HB101 as described above.Cells are plated onto LB-amp plates and incubated as described. Oneputative positive colony is inoculated into LB-amp media and DNA isisolated essentially as described. The structure of this new subclonepBS-PR1013Eco Pst Xho is verified by restriction analysis and DNAsequencing. FIG. 6 shows the construction of this plasmid.

Example 25 Preparation of pCIB270

Plasmid pBI101.3 (Catalog no. 6024.1) is obtained from Clonetech Labs,Palo Alto, Calif. This plasmid is first digested with BamHI and thenwith Salt. The plasmid pBS-PR1013Eco Pst Xho is digested with PstI. APstI/BamHI adaptor having the sequence

5′-GGGATCCCTGCA-3′ (SEQ ID No. 54)

is prepared as described in Example 5 and ligated to the PstI- digestedpBS-PR1013Eco Pst Xho. The resulting material is first digested withBamHI and then with XhoI. The BamHI/XhoI fragment is isolated, mixedwith and ligated to the digested pBI101.3, and transformed. A putativepositive clone of pCIB270 is isolated and verified by restriction andDNA sequence analysis. The preparation of this plasmid is shown in FIG.7.

Example 26 Preparation of M13mp18- or mp19-PR1013Eco Pst Xho

The plasmid pBS-PR1013Eco Pst Xho is digested with PstI and Asp718. The1.1 kb Asp718/PstI fragment is isolated after electrophoresis on a 1%TAE low-gelling agarose gel. The replicative form (RF) of the coli phageM13mp18 or M13mp19 is prepared according to Messing, J., Meth. Enymol.101: 20 (1983). Alternatively, these vectors can be obtained fromBethesda Research Labs., Gaithersburg, Md. Either vector is digestedfirst with Asp718 and then with PstI. After removal of the polylinkerpiece by electrophoresis on 0.7% TAE low-gelling agarose, the resultingvector RF is mixed and ligated in agarose with the 1.1 kb fragmentprepared above. The DNA is transformed, putative positive plaques areisolated and their structure is verified by analysis of the RF DNA. Theconstructions of M13mp18- and mp19-PR1013Eco Pst Xho are shown in FIG.8.

Example 27 Preparation of M13mp18- or mp19-PR1013Eco Pst Xho.Nco andpCIB 268

Single stranded M13mp18-PR1013Eco Pst Xho phage DNA is isolatedaccording to Messing, supra. An 18 bp oligonucleotide primer of thesequence

5′-AATCCCATGGCTATAGGA-3′ (SEQ ID No. 55)

is synthesized as described above. The primer is phosphorylated with T4polynucleotide kinase (Maniatis, T. et al., supra at p. 125) usingunlabeled ATP. After incubation at 37° C. for 60 minutes the reaction isheated to 68° C. for 15 minutes and then placed on ice. This primer andthe M13 -40 forward sequencing primer (New England Biolabs #1212) areannealed to single stranded M13mp18-PR1013Eco Pst Xho DNA after mixingin a 1:1:1 molar ratio, by slow cooling after heating for 10 minutes at68° C. The annealed mixture is placed on ice and {fraction (1/10)}volume of 10×elongation buffer (20 mM Tris buffer pH 7.5, 6 mM MgCl₂, 1mM DTT, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, and 1 mM dTTP) is added. 10units of DNA Polymerase I large fragment (Klenow fragment, New EnglandBiolabs #210) and 0.1 unit of T4 DNA ligase (NEB #202) are added and thereaction is allowed to proceed 14 hours at 15° C. At that time anadditional aliquot of each enzyme is added and the reaction is carriedout for an additional 2 hours at 25° C. before the mixture is used totransform competent JM101 E. coli. A general flow diagram of thisprocedure is presented in FIG. 9.

To identify the mutated phage, the plaques are lifted to Gene ScreenPlus (DuPont) and processed according to the manufacturer'srecommendations. The filters are pre-hybridized for four hours, andhybridized overnight at 42° C., in the manufacturer's hybridizationsolution containing 0.9 M NaCl. The filters are then sequentially washedat a NaCl concentration of 0.9 M and monitored by autoradiographyincreasing the wash temperature by 5° C. at each step. Phages identifiedby hybridization to have been mutated are sequenced to confirm the basechange.

The replicative form of one such candidate (M13mp18-PR1013Eco PstXho.Nco) is isolated and digested with PstI and BstEII to produce a 394bp fragment which is used to replace the corresponding non-mutated 394bp fragment in pBS-PR1013Eco Pst Xho as illustrated in FIG. 10. Thisresults in the preparation of pCIB268 which has an NcoI site at position930 followed by 217 bp of the coding sequence for the PR-1a gene. Thestructure of this plasmid is confirmed by sequence analysis.

Example 28 Preparation of Chimeric Genes Containing GUS and VariableLengths of the PR Promoter Sequence A. Construction of pCIB269

A fragment of 930 bp is isolated after electrophoresis of XhoI and NcoIdigested pCIB268 on a 1.0% low gelling temperature agarose TAE gel. Thisfragment is then ligated into pBS-GUS1.2 (see part B below) which hadbeen digested with XhoI and NcoI. Transformants are isolated andanalyzed by restriction analysis and DNA sequencing. One positiveplasmid is chosen and named pCIB269. The construction of this plasmid isshown in FIG. 11.

B. Construction of pBS-GUS1.2

pBS-GUS1.2 is created by three part ligation of a 391 bp SalI/SnabIfragment from pRAJ265 (Jefferson, R. A. et al., EMBO J. 6: 3091-3907(1987) and GUS User Manual, Clonetech Labs., Palo Alto, Calif.) with a1707 bp SnabI/EcoRI fragment from pBI221 (Jefferson, R. A. et al., EMBOJ. supra) and pBS digested with SalI and EcoRI (see FIG. 12).Transformants are isolated and analyzed by restriction digestion and DNAsequencing. One verified clone is named pBS-GUS1.2.

C. Construction of pCIB200

TJS75Kan is first created by digestion of pTJS75 (Schmidhauser andHelinski, J. Bacteriol. 164: 446-455 (1985)) with NarI to excise thetetracycline gene, followed by insertion of an AccI fragment from pUC4K(Messing, J. and Viena, J., Gene 19: 259-268 (1982)) carrying a NptIgene. pCIB 200 is then made by ligating XhoI linkers to the EcoRVfragment of pCIB7 (containing the left and right T-DNA borders, a plantselectable nos/nptII chimeric gene and the pUC polylinker, Rothstein, S.J. et al., Gene 53: 153-161 (1987)) and cloning XhoI digested fragmentinto SalI digested TJS75Kan.

D. Construction of pCIB271

The plasmid pCIB269 (part A above) is digested with XhoI and EcoRI andthe 3023 bp fragment carrying the PR-1a promoter linked to the GUS genewith a nos 3′ end is isolated following electrophoresis on a 1.0% lowgelling agarose TAE gel. This fragment is then ligated into a broad hostrange vector pCIB200 which has been digested with SalI and EcoRI.Transformants are isolated and analyzed by restriction analysis. Oneverified clone is named pCIB271. The construction of this clone is shownin FIG. 13.

E. Construction of pCIB272

pCIB268 (Example 27) is digested with AluI plus NcoI and an 833 bpfragment is isolated after electrophoresis on a 1×TAE 0.7% agarose gel.This fragment is ligated to the β-glucuronidase gene with the nos 3′ endin pBS-GUS1.2 (part B above). The promoter and GUS are then excised fromthis plasmid named pCIB282 as an Asp718I/BamHII fragment and ligatedinto pCIB200 which has been digested with Asp718I and BamHI beforetransformation into E. coli strain DH5. Transformants are isolated andanalyzed by restriction analysis. One verified clone is named pCIB272.

F. Construction of pCIB273

pCIB268 is digested with HaeIII plus NcoI and a 603 bp fragment isisolated after electrophoresis on a 1×TAE 0.7% agarose gel. Thisfragment is placed in front of the β-glucuronidase gene with the nos 3′end in pBS-GUS1.2. The promoter and GUS are then excised from thisplasmid named pCIB283 as an Asp718I/BamHI fragment and ligated intopCIB200 which has been digested with Asp718I and BamHI beforetransformation into E. coli strain DH5. Transformants are isolated andanalyzed by restriction analysis. One verified clone is named pCIB273.

Example 29 Preparation of a Chimeric Gene in a Hygromycin Vector

pCIB269 is digested with XhoI and EcoRI and the 3023 bp fragmentcarrying the PR-1a promoter linked to the GUS gene with a nos 3′ end isisolated following electrophoresis on a 1.0% low gelling agarose TAEgel. This fragment is then converted to a XhoI/SalI fragment by ligationwith a EcoRI/SalI adapter having the sequence

5′-AATTCGTCGACG-3′.

The XhoI/SalI fragment is ligated into SalI digested pCIB712 (Rothstein,S. J. et al., supra) to create pCIB219, see FIG. 14.

Example 30 Preparation of pCIB200/PR1-BT A. Ligation with pCIB200

The plasmid pCIB1004 (see below) is digested with SphI, phenolextracted, ethanol precpitated and resuspended. An oligonucleotide withthe sequence

5′-CCGGTACCGGCATG-3′ (SEQ ID No. 56)

is synthesized, purified, kinased, and ligated to the SphI digestedplasmid pCIB1004. After ligation, the ligase is inactivated and the DNAdigested with KpnI. This DNA is run on a 0.8% low gelling temperatureagarose gel. The plasmid pCIB200 Example 28, part C) is digested withKpnI and treated with calf-intestial alkaine phosphatase and run on a0.8% low gelling temperature agarose gel. The band containing thepCIB200 vector and the band containing the PR-1 5′ flanking sequence/BTfusion are mixed and the DNA ligated to form the plasmid pCIB200/PR1-BT.

B. Preparation of pCIB1004

The plasmid pCIB10/35Bt(607) is digested with NcoI and BamHI and run ona 0.8% low gelling temperature agarose gel. The plasmid pCIB269 (Example28, part A) is digested with NcoI and XboI and run on a 0.8% low gellingtemperature agarose gel. The plasmid pCIB710 (Rothstein, S. J. et al.,supra) is digested with SalI and BamHI and run on a 0.8% low gellingtemperature agarose gel. The band with the SalI and BamHI digestedvector containing a CaMV 35S promoter 3′ end cloned into pUC19, the bandcontaining the BT gene and the band containing the PR-1 5′ flankingregion are excised, mixed and the DNA ligated to form the plasmidpCIB1004.

C. Preparation of pCIB10/35Bt(607)

pCIB10/35Bt(607), a deleted protoxin gene containing a sequence codingfor approximately 607 amino acids, is prepared from plasmidpCIB10/35Sbt, a plasmid containing the protoxin gene from Bacillusthuringiensis endotoxin. E. coli MC1061 containing pCIB10/35Sbt wasdeposited at the American Type Culture Collection, ATCC No. 67329, Feb.27, 1987. A deleted protoxin gene is made by introducing a BamHIcleavage site (GGATCC) following nucleotide 1976 in the BT genesequence. This is done by cloning the BamHI fragment containing theprotoxin sequence from pCIB10/35Sbt into mp18, and using standardoligonucleotide mutagenesis procedures. After mutagenesis,double-standard replicative form DNA is prepared from the M13 clone,which is then digested with BamHI. The approximately 1.9 kb fragmentcontaining the deleted protoxin gene is inserted into BamHI-cleavedpCIB10/710. The resulting plasmid is selected for on kanamycin. Adetailed description for the preparation is given in U.S. patentapplication Ser. No. 122,109, filed Nov. 18, 1987, which is incorporatedby reference herein in its entirety.

Example 31 Preparation of pCIB1233 (pCIB200/PR1-AHAS) A. Ligation withpCIB200

The plasmid pCIB200 is digested with KpnI and XbaI and run on a 0.8% lowgelling temperature agarose gel. The plasmid pCIB1216 is digested withKpnI and XbaI and run on a 0.8% low gelling temperature agarose gel. The˜4.3 kb band containing the PR-1 5′ flanking sequence fused to the AHAScoding sequence and the band containing the pCIB200 vector are excised,mixed and the DNA ligated to form pCIB1233.

B. Preparation of pCIB1216

The plasmid pCIB269 (Example 28, part A) is digested with XbaI and NcoIand run on a 0.8% low gelling temperature agarose gel. The plasmidpCIB1207 (see below) is digested with XbaI and NcoI and run on a lowgelling temperature agarose gel. The band containing the bluescriptvector with the PR-1 5′ flanking sequence and the 3.3 kb band containingthe AHAS coding sequence are excised, mixed and the DNA ligated to formthe plasmid pCIB1216.

C. Preparation of Plasmid pCIB1207 Containing the Arabidopsis AHAS Gene

Total plant DNA is isolated from 5 weeks old Arabidopsis thalianaecotype Columbia. 250 g of this DNA is digested with 4000 units of therestriction enzyme XbaI for 4 hours. The digest is fractionated byelectrophoresis on a 0.8% agarose gel. The DNA fragments between 6.5 kband 4.36 kb (based upon HindIII-digested lambda DNA size markers) areisolated from the gel using NA-45 DEAE membrane (Schleicher and Schuell,Keene, N.H., USA) following the manufacturer's recommended procedures.XbaI digested and phosphatase-treated lambda ongC (longC) arepreparation is obtained from Stratagene Cloning Systems (La Jolla,Calif., USA). 0.1 g of the Arabidopsis XbaI insert DNA is ligated to 1.0g of the longC arms according to procedures recommended by Stratagene.2.5 l of the ligation reaction is packaged in lambda phage heads usingthe Gigapack Plus kit (Stratagene Cloning Systems) following proceduresrecommended by the manufacturer. The activity of the library is titeredon E. coli VCS257 (Stratagene Cloning Systems).

50 g of pE16/8-c4 plasmid DNA (J. Polaina, Carlsberg Res. Comm. 49,577-584) are digested with EcoRI, and the digest products are resolvedon a 0.8% agarose gel. The 2 kb EcoRI fragment containing the codingsequence for the yeast AHAS gene is isolated from the gel using NA45DEAE membrane following procedures recommended by the manufacturer.Radiolabeled probes to the 2 kb fragment are synthesized on the day ofthe hybridization reaction with the random priming reaction using PrimeTime kit (International Biotechnologies Inc., New Haven, Conn., USA),following procedures recommended by the manufacturer.

250,000 recombinant phages are plated on VCS257. Duplicate membranelifts of the phage plaques are made using Colony/Plaque Screen membranes(NEN Research Products, Du Pont, Boston, Mass., USA) followingprocedures recommended by the manufacturer. The membranes areprehybridzed in LS hybridization buffer, then transferred to 150 ml offresh LS hybridization buffer containing 0.25 g of ³²P-labelled yeastAHAS gene probe prepared as described above. The membranes are incubatedfor 16 hours at 42° C. with gentle shaking, washed twice at roomtemperature with 2×SSC for 15 minutes per wash, washed three times at42° C. in LS wash buffer (25% formamide, 5×SSC, 1% SDS) for 2 hours perwash, and washed twice at in rinse buffer (0.1×SSC, 0.1% SDS) for 30minutes per wash. The membranes are mounted and fluorographed withCronex Lightening Plus Sceens (Du Pont, Wilmington, Del., USA) and XAR-5film (Kodak). The plaques from regions of the plates that gave positivefilm signals on both membrane lifts are picked and replated at a lowdensity of 300 plaques per 150 mm plate, and the screening process isrepeated until single hybridizing plaques are isolated.

Miniprep of phage DNA from the plaque-purified phage is carried outfollowing procedures accompanying the LambdaSorb Phage Adsorbent kit(Promega, Madison, Wis., USA). The phage DNA is cut with restrictionenzyme XbaI and the 5.8 kb insert fragment is cloned into the XbaI siteof pBS(−) plasmid (Stratagene Cloning Systems). The identity of thecloned fragment with the XbaI fragment containing the Arabidopsis AHASgene is verified by comparing its restriction map and DNA sequence withthose published for the gene by Haughn et al., Mol. Gen. Genet. 211: 266(1988), and Mazur et al., Plant Physiol. 85: 1110 (1987). This plasmidis designated pCIB1207.

Example 32 Preparation of pCIB1232 (pCIB200/PR1-AHAS-SuR) A. Ligationwith pCIB200

The plasmid pCIB200 (Example 28, part C) is digested with KpnI and XbaIand run on a 0.8% low gelling temperature agarose gel. The plasmidpCIB1230 is digested with KpnI and XbaI and run on a 0.8% low gellingtemperature agarose gel. The ˜4.3 kb band containing the PR-1 5′flanking seqeunce fused to the AHAS-SuR coding sequence and the bandcontaining the pCIB200 vector are excised, mixed and the DNA ligated toform pCIB1232.

B. Preparation of pCIB1230

Plasmid pCIB1208 (see below) is digested with XbaI and NcoI and thefragments are separated by electrophoresis on a 1×TAE 0.8% low gellingtemperature agarose gel. The plasmid pCIB269 (Example 28, part A) isdigested with NcoI and XbaI and run on a 1×TAE 0.8% low gellingtemperature agarose gel. The pCIB269 fragment containing the bluescriptvector and the PR-1 promoter and the 3.3 kb fragment containing themutated AHAS gene (identified by cross homology and by restrictionfragment analysis with the gene descibed Mazur et al., Plant Physiol.85: 1110-1117 (1987)) are excised, melted and ligated together to createa clone designated as pCIB1230.

C. Preparation of pCIB1208

A genomic DNA library is constructed as described in Example 31, part Cusing DNA isolated from Arabidopsis plants selected for resistance tosulfonylurea herbicide as described by Haughn and Somerville, Mol. Gen.Genet. 204: 430-434 (1986). Plaques containing genes encodingacetohydroxy acid synthase (AHAS) are identified by cross hybridizationwith a yeast acetohydroxy acid synthase gene probe (see Example 31, partC). The recombinant DNA is subcloned into Bluescript (Stratagene)creating a clone designated as pCIB1208. This plasmid contains a geneencoding a mutated acetohydroxyacid synthase that is resistant toinhibition by sulfonylurea herbicides.

Example 33 Isolation of a Genomic Clone Encoding the Basic Form ofβ-1,3-Glucanase (Glucan Endo-1,3-β-glucosidase) from Nicotiana Tabacum

High molecular weight DNA is prepared from leaves of Nicotiana tabacumcv. Havana 425 by the CETAB procedure (Murray and Thompson, Nucleic AcidRes. 8: 4321 (1980)). 100 g of DNA is digested to completion with SacI.This DNA is separated by electrophoresis on a 0.8% agarose gel. Theregion of the gel corresponding to a molecular weight range of 3 to 7 kbis cut into 6 equally sized strips and the DNA is electroeluted fromthese strips and precipitated as separate fractions. The DNA isresuspended and an aliquot from each fraction is run on an agarose geland analyzed by Southern blotting. The fraction that contains DNA whichhybridizes to a β-1,3-glucanase cDNA probe (Shinshi, H. et at., Proc.Natl. Acad. Sci. USA 85: 5541-5545 (1988)) is pooled and used inconstructing libraries.

The cloning vector lambdaOngC purchased from Stratagene Corp. isdigested with Sacl. 1 g of this DNA is mixed with 0.1 g of the SacIdigested tobacco DNA, and the DNA is ligated with T4 DNA ligaseaccording to the manufacturer's suggestions. The ligated DNA is thenpackaged into phage particles using an in vitro packaging procedureaccording to Stratagene. The phages are plated with bacteria assuggested in the lambda manual. About 75,000 plaques are screened usinga 32-P labeled β-1,3-glucanase cDNA probe. 11 positive plaques areidentified. These plaques are purified by successive rounds of platingand screening.

DNA is isolated from the purified clones and the clones are analyzed byrestriction digestion using HindIII, EcoRI, and SacI. The clones are oftwo types, one represented by the clone 39.1 and the other representedby the clone 39.3. The 4.5 and 4.7 kb inserts in clone 39.1 and 39.3,respectively, are subcloned into the bluescript plasmid digested withSacI, and the subclones pBSGluc39.1 (SacI fragment derived from thelambda clone 39.1) and pBSGluc39.3 (SacI fragment derived from thelambda clone 39.3) are isolated. The sequence of the DNA in the SacIfragments contained in the subclones pBSGluc39.1 and pBSGluc39.3 isdetermined by DNA sequencing and shown in SEQ ID Nos. 5 and 6,respectively. The coding sequence is found and a large interveningsequence is located near the 5′ end of the coding sequence.

Example 34 Identification of the Transcriptional Start Site for theβ-1,3-Glucanase Gene A. Primer Extension Mapping

A synthetic DNA primer is synthesized which is complementary to bases2399 to 2416 and used in primer extension experiments as described inExample 6. The RNA for these experiments is isolated from Phytophthoraparasitica var. nicotiana infected tobacco. The primer extensionproducts are run against a molecular weight standard in which thelabeled pimer is used in dideoxy DNA sequencing reactions with thesubclone pBSGluc39.1 used as a template. Extension products areidentified, after gel electrophoresis and autoradiography as describedin Example 6, that correspond to positions 1432, 1446 and 1447 of theβ-1,3-glucanase 39.1 sequence, SEQ ID No. 5. Since a large intron existsbetween positions 1554 and 2345 of the pBSGluc39.1 sequence, themolecular weight ladder might not reflect the correct molecular weightof the extension products. Therefore, a second primer extension mappingexperiment is conducted using a primer that is complementrary topositions 1530 to 1547. Using this primer, three 5′ ends of theglucanase mRNA are mapped to A residues at positions 1434, 1448 and1449.

B. S1 Nuclease Mapping

A synthetic oligonucleotide complementary to positions 1415 to 1504 issynthesized for use as a probe in S1 nuclease mapping. Theoligonucleotide is kinased at the 5′ end using 32P-ATP and T4polynucleotide kinase according to the supplier's recommendation.Following phenol extraction and ethanol precipitation the labeledoligonucleotide is resuspended in formamide hybridization buffer (seeExample 7) at a concentration of about 2 nM. The RNA used in theseexperiments is isolated from Phytophthora parasitica infected tobacco asin the previous example. S1 mapping is conducted on this RNA using thelabeled oligonucleotide as described in Example 7. After gelelectrophoresis three bands are detected that correspond to positions1434, 1448 and 1449 on the pBSGluc39.1 DNA sequence.

C. Determination of the Transcriptional Start Site

Primer extension and S1 nuclease mapping procedures both place the 5′ends of the mRNA at positions 1434, 1448 and 1449. Therefore thesesites, which are all adenine residues, correspond to the transcriptionalstart site of the β-1,3-glucanase gene.

Example 35 Preparation of pBSGluc39.1/GUS

Oligonucleotides with the sequences

A) 5′-GTTTATGTGAGGTAGCCATGGTGGGAAGACTTGTTGGG-3′ (SEQ ID No. 57); and

B) 5′-GATCGCGGTACCGAGCTCCTCTAGGGGGCCAAGG-3′ (SEQ ID No. 58)

are synthesized, purified and used in a polymerase catalyzed reaction(PCR) according to the supplier's directions (Perkin Elmer Cetus, DNAthermal cycler; and Perkin Elmer Cetus, GeneAmp Reagent Kit) to amplifya 1494 bp fragment of DNA from pBSGluc39.1. The oligos are designed toadd a KpnI site immediately 5′ to the SacI site at base-pair 1 of the39.1 glucanase sequence and to generate a new NcoI site at base-pair1462. The DNA derived from the PCR amplification procedure isphenol/chloroform extracted and ethanol precipitated. The DNA isresuspended and digested with both NcoI and KpnI and run on a 0.8%low-gelling temperature agarose gel. The 1462 bp band is excised andused in the following ligation. The plasmid pBS-GUS1.2 (Example 28, partB) is digested with NcoI and KpnI and run on a 0.8% agarose gel and theband containing the vector is excised. The band containing thepBS-GUS1.2 vector and the 1462 bp band from the PCR reaction are ligatedand used to transform E. coli. Positive colonies are picked and screenedfor those that contain the 1462 bp insert. The plasmids containinginserts are putative clones of pBSGluc39.1/GUS, however, since themutation rate in this procedure is relatively high (˜{fraction (1/1000)}bases), several clones have to be sequenced throughout the 1462 bpfragment. One clone which has the expected sequence is chosen as theclone pBSGluc39.1/GUS.

Example 36 Preparation of pBSGluc39.3/GUS

Oligonucleotides with the sequences

A) 5′-GTTTATCTGAGGTAGCCATGGTGAGAAGACTTGTTGGA-3′ (SEQ ID No. 59); and

B) 5′-GATCGCGGTACCGAGCTCCCTTGGGGGGCAAG-3′ (SEQ ID No. 60)

are synthesized, purified and used in a PCR reaction to amplify a 1677bp fragment from pBSGluc39.3. The oligonucleotides are designed tointroduce a new KpnI site immediately adjacent to the SacI site atposition 1 of the glucanase 39.3 sequence and a new NcoI site atposition 1646. The DNA from the PCR amplification is phenol/chloroformextracted, ethanol precipitated, resuspended, digested with both NcoIand KpnI and run on a 0.8% low-gelling temperature agarose gel. The bandat 1646 bp is excised and used in the following ligation. pBS-GUS1.2 isdigested with NcoI and KpnI and run on a 0.8% low gelling temperatureagarose gel. The band containing the vector is excised, mixed with the1646 bp band from the PCR reaction and ligated to form the plasmidpBSGluc39.3/GUS. As before, several putative plasmids are isolated andseqeunced and one with the expected sequence is picked as the plasmidpBSGluc39.3/GUS.

Example 37 Preparation pCIB200/Gluc39.1-GUS and pCIB200/Gluc39.3-GUS

A. pCIB200 (Example 28, part C) is digested with KpnI and XbaI and runon a 0.8% low gelling temperature agarose gel. The plasmidpBSGluc39.1/GUS (Example 35) is digested with KpnI and XbaI and thefragments are separated on a 0.8% low gelling temperature gel. The bandscontaining the pCIB200 vector and the KpnI-XbaI band containing the 5′flanking sequence of the glucanase 39.1 gene fused to the GUS gene areexcised and the DNA ligated to form the plasmid pCIB200/Gluc39.1-GUS.

B. Likewise, plasmid pBSGluc39.3/GUS (Example 36) is digested andligated with digested pCIB200 to form the plasmid pCIB200/Gluc39.3-GUS.

Example 38 Preparation of pCIB200/Gluc39.1-BT and pCIB200/Gluc39.3-BT A.pCIB200/Gluc39.1-BT

The plasmid pBSGluc39.1/GUS is digested with KpnI and NcoI and run on a0.8% low gelling temperature agarose gel. The plasmid pCIBl1004 (Example30, part B) is digested with KpnI and NcoI and run on a 0.8% low gellingtemperature agarose gel. The plasmid pCIB200 (Example 28, part C) isdigested with KpnI, dephosphorylated using calf-intestine alkalinephosphatase and run on a 0.8% low gelling temperature agarose gel. Theband containing the pCIB200 vector, the band containing the glucanase 5′flanking region, and the band containing the BT gene are excised, mixedtogether and ligated to form the plasmid pCIB200/Gluc39.1-BT.

B. pCIB200/Gluc39.3-BT

Likewise, plasmid pBSGluc39.3/GUS is digested and ligated with digestedpCIB1004 and digested pCIB200 to form the plasmid pCIB200/Glu39.3-BT.

Example 39 Preparation of pCIB200/Gluc39.1-AHAS andpCIB200/Gluc39.3-AHAS A. Ligation with pCIB200

The plasmid pBSGluc39.1/AHAS is digested with KpnI and XbaI and run on a0.8% low gelling temperature agarose gel. The plasmid pCIB200 (Example28, part C) is digested with KpnI and XbaI and run on a 0.8% low gellingtemperature agarose gel. The band containing the glucanase/AHAS fusionand the band containing the pCIB200 expression vector are excised, mixedand the DNA ligated to form the plasmid pCIB200/Gluc39.1-AHAS.

Likewise, plasmid pBSGlu39.3/AHAS is digested and ligated with digestedpCIB200 to form the plasmid pCIB200/Glu39.3-AHAS.

B. Preparation of Plasmids pBSGlu39.1/AHAS and pBSGlu39.3/AHAS

The plasmid pBSGluc39.1/GUS (Example 35) is digested with NcoI and XbaIand run on a 0.8% low gelling temperature agarose gel. The plasmidpCIB1207 (Example 31, part C) is digested with NcoI and XbaI and run ona 0.8% low gelling temperature agarose gel. The band containing theglucanase 5′ flanking sequence and vector and the band containing theAHAS coding sequence are excised, mixed and the DNA ligated to producethe plasmid pBSGluc39.1/AHAS.

Likewise, plasmid pBSGlu39.3/GUS (Example 36) is digested and ligatedwith digested pCIB1207 to form the plasmid pBSGlu39.3/AHAS.

Example 40 Preparation of pCIB200/Gluc39.1-AHAS-SuR andpCIB200/Gluc39.3-AHAS-SuR A. Ligation with pCIB200

The plasmid pBSGluc39.1/AHAS-SuR is digested with KpnI and XbaI and runon a 0.8% low gelling temperature agarose gel. The plasmid pCIB200(Example 28, part C) is digested with KpnI and XbaI and run on a 0.8%low gelling temperature agarose gel. The band containing theglucanase/AHAS-SuR fusion and the band containing the pCIB200 expressionvector are excised, mixed and the DNA ligated to form the plasmidpCIB200/Gluc39.1-AHAS-SuR.

Likewise, plasmid pBSGluc39.3/AHAS-SuR is digested and ligated withdigested pCIB200 to form the plasmid pCIB200/Gluc39.1-AHAS-SuR.

B. Preparation of Plasmids pBSGluc39.1/AHAS-SuR and pBSGluc39.3/AHAS-SuR

The plasmids pBSGluc39.1/GUS (Example 35) and pBSGluc39.3/GUS (Example36) are digested with NcoI and XbaI and the restriction fragments areseparated on a 1×TAE 0.8% low gelling temperature agarose gel. pCIB1208(Example 32, part C) is digested with XbaI and NcoI and the 3.3 kbfragment containing the mutated AHAS gene (sulfonylurea herbicideresistant, SuR) is isolated by agarose gel electrophoresis. Thefragments containing the glucuronidase promoter and pBS vector areexcised, melted and ligated with the AHAS fragment to create clonesdesignated pBSGluc39.1/AHAS-SuR and pBSGluc39.3/AHAS-SuR, respectively.

Example 40A Cloning of cDNAs Corresponding to SAR CHX-independent GenesFrom Tobacco

A number of cDNAs were cloned by differential screening from cDNAprepared from induced and non-induced tissue. The induced cDNA wasprepared from tobacco leaves which had been pre-treated with methylbenzo-1,2,3-thiadiazole-7-carboxylate (BTH), whereas the non-inducedcDNA was prepared from tissue which had not been pre-treated with BTH.cDNA libraries were prepared in λZAP II (STRATAGENE). A standarddifferential screening technique was used. Plaques carrying induced cDNAwere plated at low density and transferred to two sets of hybridizationfilters. Known SAR gene sequences (from previous cloning experiments)were hybridized to the first filter and uninduced cDNA to the second.The second filter was then stripped and hybridised with induced cDNA.Plaques which hybridized with the induced cDNA probe, but not with theuninduced cDNA probe or with known SAR gene sequences were potentialnovel SAR genes and were picked directly for further analysis. Platinghad been at a plaque density which was sufficiently low to enable theseplaques to be picked as nearly pure plaques. Multiple candidates foreach plaque were in vivo excised, according to the manufacturter'srecommended conditions, for further screening by Northern hybridizationto RNA isolated from either untreated or BTH-treated tobacco plants.Individual clones chosen from the secondary screen were further analyzedby Northern hybridisation to RNA isolated from tobacco plants which hadbeen pre-treated with salicylic acid (3 mM), INA (1 mM), and BTH (1mg/ml), all in the presence or absence of cycloheximide (CHX; 1 mg/ml).Inducer pre-treatments were done at 2 h, one day, and eight days beforethe isolation of RNA, whereas CHX treatment was done at one day beforeisolation of RNA. Four cDNAs were found induced in a protein synthesisindependent fashion. The genes corresponding to these cDNAs have beendesignated 1.1.1, 11.3.8, 11.30.13, and 1.4.3 (see SEQ. ID Nos. 99-103)and are likely signal transducers of the SAR response. Gene 1.4.3 haspreviously been disclosed as a thioredoxin (Brugidou et al., Mol. Gen.Genet. 238: 285-293 (1993)). However, this is the first disclosure ofthe gene's likely involvement in the systemic acquired response. TwocDNAs were found to be expressed in a protein synthesis dependentfashion. These were designated 66B1 and 14.22.3 and are listed as SEQ.ID Nos. 104 and 105.

Example 40B Cloning of cDNAs Corresponding to SAR CHX-independent GenesFrom Arabidopsis

Total RNA was isolated from the following Arabidopsis lines: (1)untreated, (2) INA treated (0.25 mg/ml), (3) CHX treated (1 mg/ml), and(4) INA & CHX. Treatments were made 1 day before RNA isolation. RNA thusisolated was was subjected to “differential display” using the protocoldescribed by Liang and Pardee, Science 257: 967-971 (1992). Amplifiedfragments which were found in both the INA as well as the INA & CHXtreated RNA samples were gel-purified as used as probes on Northernblots carrying similarly induced RNA samples. Fragments for whichNorthem hybridization confirmed the induction profile apparent fromdifferential display were subcloned into a plasmid vector. Using thecloned fragment it was possible to isolate near full-length cDNAs from acDNA library produced by BTH induction (see Example 54). The cDNA cDPA2was cloned using this technique and is induced by the SAR response in aprotein synthesis independent fashion. Its LA sequence is listed in Seq.ID No. 106.

E. ISOLATION OF NOVEL cDNA CLONES ENCODING PR PROTEINS

This group of Examples describes the isolation of novel cDNA clonesencoding plant PR proteins. It is divided into 3 sections: Section Acovers the construction of libraries used in the isolation of clones.Section B covers the identification and purification of clones thusisolated. Section C covers the development of a novel cDNA cloningtechnology. We have found that cDNAs isolated from uninoculated tissueof inoculated plants express messenger RNAs that encode proteins whichare involved in manifestation of the plant's systemic acquiredresistance to a variety of plant pathogens, such as bacteria, virusesand fungi. These are distinguished from those mRNAs expressed ininoculated tissue, which are often related to generalized stressconditions, such as pathogen attack and cell death. We also have foundthat this same set of systemic acquired resistance genes is alsoinvolved in the expression of proteins in chemically induced resistance.

1. Construction of cDNA Libraries

Example 41 Preparation of cDNA Library From TMV-Infected Tobacco Leaves

Nicotiana tabacum cv. Xanthi-nc leaves are infected with tobacco mosaicvirus and harvested five days post-infection. Total RNA is prepared asdescribed above and poly A+ RNA is isolated using standard techniques. AcDNA library is constructed using this poly A+ RNA in the Lambda ongCcloning vector (Stratagene) essentially as described (Gubler, U. andHoffman, B. J., Gene 25: 263 (1983)).

Example 42 Preparation of a cDNA Library From Uninfected Leaves of TMVInoculated Tobacco Using a Novel cDNA Cloning Vector

pCGN1703, a plasmid cDNA cloning vector of the vector-primer typedescribed by Okayama and Berg, Mol. and Cell Biol. 2:161-170 (1982), isconstructed as an improved vector to simplify the cloning process andallow easy shuttling of libraries into a phage vector, as well as toprovide additional functions that are, outside the present use.

A. Construction of the pCGN1703 Cloning Vector

Bluescribe M13- (Stratagene, Inc.) is used as a starting plasmid. TheBamHI site is deleted by BamHI digestion and mungbean nucleasetreatment, followed by ligation with T4 DNA ligase to yield pCGN1700.This plasmid is digested with EcoRI and SacI and then ligated with adouble stranded synthetic polylinker created by annealing twooligonucleotides of the sequence

[oligo #47] 5′-AATTTCCCGGGCCCTCTAGACTGCAGTGGATCCGAGCT-3′ (SEQ ID No. 61)

[oligo #46] 5′-CCGATCCACTGCAGTCTAGAGGGCCCGGGA-3′ (SEQ ID No. 62)

The resulting plasmid thus has additional restriction sites for SmaI,ApaI, XbaI, PstI, and BamHI, and is designated pCGN1702. Note that theEcoRI site is not reconstructed. pCGN1702 is digested to completion withHindIII and made blunt ended with T4 polymerase. The resultant DNA issubjected to partial digestion with PvuII and then ligated with T4 DNAligase. A transformant is selected that had deleted the 214 bpHindIII-PvuII fragment which included the lac operator-promoter region;this plasmid is designated pCGN1703. Use of vector-primer plasmids suchas pCGN1703 is described previously (1). Alexander, Methods in Emymology154:41-64(1987)). As described in the ADDENDUM section of that work, thepresent vector is a monomer vector. The T-tracts used to prime cDNAsynthesis are present on both ends of the vector during the reversetranscriptase and terminal transferase (G-tailing) reactions, and thelinker DNA used to circularize the final products of cDNA cloning withpCGN1703 have the generalized structure as follows: T7 promoter, amultiple cloning site (SmaI, ApaI, XbaI, PstI, BamHI, NotI, EcoRI,SacI), C:G homopolymer tract (10-15 residues), cDNA insert (5′-3′ ofmRNA-sense strand), A:T homopolymer tract (40-100 residues), and anothermultiple cloning site (KpnI, SmaI, XbaI, SalI, PstI and SphI), allcontained within the plasmid backbone derived from Bluescribe M13- asdescribed above.

B. Construction of the cDNA Library Using pCGN1703

Xanthi.nc tobacco plants are grown in a phytotron until they areapproximately 10-12 inches tall. Two leaves near the bottom of eachplant are inoculated with either a mock buffer-only sample (10 mM sodiumphosphate, pH7.0) or a sample of TMV (10 g/ml in the same buffer).Eleven days later 3-4 upper leaves, which had not been inoculated, areharvested and frozen in liquid nitrogen. Poly-A+ mRNA is isolated bymethods previously described (Hall et al., PNAS USA 75: 3196-3200(1978); Maniatis et al., “Molecular Cloning”, p. 297-209 (1982)). A cDNAlibrary is constructed, using the poly-A+ RNA isolated from TMV-inducedleaves, in the cDNA vector pCGN1703 by methods previously described (D.Alexander, Methods in Enzymology 154:41-64 (1987)).

Plasmid DNA of the amplified cDNA library (D. C. Alexander, 1987, supra)is digested to completion with EcoRI and sub-cloned into the EcoRI siteof gt-10 (Stratagene, Inc.). Note that the plasmid vector remainsattached to the cDNAs and is also cloned into the phage vector.Therefore, using this process, two cDNA libraries are constructed, onein which the library is contained in a plasmid vector and the other inwhich the cDNA library is contained in a phage vector.

Example 43 Preparation of a cDNA Library From TNV Infected CucumberLeaves

Cucumber leaves are infected with Tobacco Necrosis Virus (TNV) and RNAis isolated 5 days after infection as described above. Poly A+ RNA isisolated by standard techniques and a cDNA library is constructed in thelambda Zap cloning vector (Stratagene), essentially as described(Gubler, U. and Hoffman, B. J., Gene 25: 263 (1983)).

2. Identification, Isolation and Characterization of cDNA ClonesEncoding PR Proteins

The Examples below describe the identification, isolation andcharacterization of cDNA clones encoding PR proteins.

Example 44 Isolation of cDNA's Encoding PR-1a, PR-1b and PR-1c

About 300,000 plaques from the cDNA library prepared above are screenedwith an oligonucleotide of the sequence:

5′CAAAACTCTCAACAAGACTATTTGGATGCCC 3′ (SEQ ID No. 63).

25 positive plaques are purified by standard techniques and DNA isprepared from the phage. A fragment of the recombinant phage whichcontains the cDNA is subcloned into the bluescipt plasmid. A partialcDNA sequence of each clone is determined by DNA sequencing. It is foundthat the 26 clones can be typed into three classes of cDNA's. Class 1 isrepresented by the clone pBSPR1-207 (see SEQ ID No. 9), class 2 isrepresented by the clone pBSPR1-1023 (see SEQ ID No. 10) and class 3 isrepresented by the clone pBSPR1-312 (see SEQ ID No. 11). In order todetermine the identity of the three clones relative to the known PR-1proteins, the amino acid sequence data for the PR-1a and PR-1b proteinsdetermined above is compared to the amino acid sequences deduced fromthe three representative cDNA clones.

When the protein sequence for PR-1a was compared to the deduced aminoacid sequence derived from pBSPR1-207 there was agreement at everyresidue. However, when the deduced amino acid sequence derived from thepBSPR1-1023 or pBSPR1-312 peptides was compared to the protein sequencefor PR-1a there were seven and six mismatches, respectively. Thus, thesequence data clearly demonstrates that the clone pBSPR1-207 encodes thePR-1a protein.

When the amino acid sequence for the PR-1b protein was compared to thededuced amino acid sequence derived from the pBSPR1-1023 protein therewas agreement at every residue. However, in comparisons to the sequencederived from pBSPR1-207 or pBSPR1-312, there were three or sixmismatches, respectively. Thus, the data clearly demonstrates that theclone pBSPR1-1023 encodes the PR-1b protein. Further, by default, theclone pBSPR1-312 is determined to encode the PR-1c protein. Thesequences of the cDNA's encoding PR-1a, PR-1b and PR-1c are included inSEQ ID Nos. 9, 10 and 11, respectively.

Example 45 Isolation of cDNA Clones Encoding PR-R Major and PR-R minor

About 300,000 plaques from the library constructed above are screenedwith an oligonucleotide probe of the sequence

5′GAACTTCCTAAAAGCTTCCCCTTTTATGCC-3′ (SEQ ID No. 64).

Fifteen positive plaques are purified by standard techniques and DNA isprepared from the phage. A fragment of the recombinant phage whichcontains the cDNA is subcloned into the bluescript plasmid. Partial DNAsequence of the cDNA insert reveals that the clones can be typed intotwo classes. The sequence of one of these clones, pBSPRR-401, whichencodes the major form of PR-R (Pierpoint, W. S. et al., Physiol. PlantPathol. 31: 291 (1987) and above) is presented in SEQ ID No. 4. Theidentity of this cDNA as encoding the major form of PR-R major isconfirmed by comparing the encoded protein sequence to the aminoterminal sequence determined experimentally above and by comparing tothe sequence presented for the PR-R major isoform by Pierpoint, et al.,supra. This encoded protein has a very strong homology to a knowntrypsin/alpha-amylase inhibitor isolated from maize (Richardson, M. etal., Nature 327: 432 (1987)). The cloned PR-R may be an inhibitor ofeither proteases or alpha-amylases and as such may confer insecticidal,viricidal or fungicidal activity to plants when expressedtransgenically.

Example 46 Isolation of cDNA Clones Encoding PR-P and PR-Q

About 300,000 plaques of the cDNA library prepared above are screenedusing a labeled cDNA probe encoding the tobacco basic chitinase gene(Shinshi et al., Proc. Natl. Acad. Sci. USA 84: 89-93 (1987)) andwashing filters at 50° C. in 0.125 mM NaCl, 1% SDS, 40 mM sodiumphosphate (pH 7.2), 1 mM EDTA. 24 positive plaques are purified and theDNA sequence of one clone named pBScht15 is determined. This sequence ispresented in SEQ ID No. 7.

The protein encoded in the clone of this sequence is determined to bethe pathogenesis-related protein Q based on: 1) limited structuralhomology to the the basic tobacco chitinase and 2) identity to theamino-terminal protein sequence of PR-Q and identity to the sequence ofa number of internal peptides derived from PR as determined by proteinsequencing (see above). The isolated clone appears to be a truncatedcDNA. In order to isolate the 5′ end of the cDNA, the end of the mRNA isfirst determined.

An oligonucleotide primer of the sequence:

5′CAGCAGCTATGAATGCAT 3′ (SEQ ID No. 65),

referred to as oligo A, is synthesized by β-cyanoethylphosphoramiditechemistry and purified by standard techniques. This primer is used in aprimer extension experiment as above using RNA isolated from TMVinfected leaves. Two extension products are visualized byautoradiography corresponding to mRNA end points that are 43 bp and 53bp longer than the pBScht15 cDNA.

In order to isolate the 5′ end of the PR-Q cDNA, a novel method ofcloning is developed based on Polymerase-catalyzed Chain Reaction (PCR)technology. Essentially, two primers are used to amplify and then clonethe 5′ end of the cDNA clone from the cDNA library. One primer (oligo A)which is complementary to the sense-strand of the cDNA and located about160 bases into the pBScht15 cDNA and a second primer (either oligo B orOligo C, below) which will prime into the cDNA from either side of thelambda cloning vector are used in this procedure.

Oligo A is complementary to the PR-Q mRNA and contains a sequencerecognized by the endonuclease NsiI. Two other oligo nucleotides withthe sequence

5′GGCAGGGATATTCTGGC 3′ (SEQ ID No. 66) and

5′ TGCAAAGCTTGCATGCC 3′ (SEQ ID No. 67)

are synthesized, purified and named oligo B and oligo C.

Oligo B has the same sequence as part of the lambda OngC cloning vectorand Oligo C is complementary to the polylinker of the Lambda OngCcloning vector.

In order to clone the 5′ end of the PR-Q cDNA, two PCR reactions arecarried out, one using oligos A and B and the other using oligos A andC. An aliquot of the cDNA library is used as a template for thereaction. The two reactions are necessary to amplify clones that hadbeen ligated into the lambda OngC vector in either direction. As acontrol, reactions are also performed on aliquots of the purified phagelysate using the chitinase 15 isolate.

After amplification, the DNA is purified and digested with NsiI andEcoRI and runs on a 1.5% LGT agarose gel. Gel slices containing DNAfragments longer than the control are excised and ligated intopBluescript which is digested with both EcoRI and PstI as describedabove.

After transformation, positive colonies are isolated and the DNA insertanalyzed by DNA sequencing. It is found that several inserts contain the5′ end of the PR-Q cDNA and others contain the 5′ end of PR-P (asdetermined by comparing the amino acid sequence deduced from the clonesto the protein sequence determined above).

The 3′ end of PR-P is then isolated from the cDNA library by screeningabout 100,000 clones of the cDNA library with a probe from one of the 5′isolates of PR-P, pBScht5′-5. Positive phage are isolated and purifiedand the insert is subcloned into pBluescript Several inserts aresequenced and one, pBSCht28, is determined to encode PR-P. The cDNAsequence of PR-P and PR-Q are shown in SEQ ID Nos. 12 and 7,respectively.

Example 47 Isolation of cDNA Clones Encoding PR-O′

About 300,000 plaques of the cDNA library described above are screenedusing a labeled cDNA probe encoding the basic form of β-1,3-glucanase(Shinshi, H. et al., Proc. Natl. Acad. Sci. USA 85: 5541-5545 1988) andwashing filters at 50° C. in 0.125 M NaCl, 1% SDS, 40 mM sodiumphosphate (pH 7.2), 1 mM EDTA. 15 positive plaques are isolated and theinsert is subcloned into the bluescript plasmid. Partial DNA sequencingreveals that pBSGL5 and pBSGL6e encode identical cDNA's which have about55% homology to the known DNA sequence of the basic form of β-1,3glucanase. The sequence of the cDNA in clone 5 and 6 is determined andshown in SEQ ID No. 13 for the cDNA in the plasmid pBSGL6e.

It is concluded that this cDNA encodes the PR-O′ protein (an acidic formof β-1,3 glucanase) based on the comparison to the amino acid sequenceof peptides derived from the PR-O′ protein. In dot matrix comparison tothe basic β-1,3-glucanase it is found that the cDNA sequence in SEQ IDNo. 13 is probably missing about 80 bases from the 5′ end.

In order to isolate the full-length form of the PR-O′ cDNA, the libraryis rescreened with a labeled EcoRI restriction fragment derived from thepBSGL6e plasmid. Six positive clones are isolated, purified andsubcloned into the bluescript plasmid. The sequence of the inserts inthe plasmids are determined by DNA sequencing. One clone, pBSGL5B-12, is87 base pairs longer than the cDNA in pBSGL6e. The sequence of this cDNAis shown in SEQ ID No. 14.

Example 48 Isolation of cDNA Clones Encoding PR-2, PR-N, PR-O, PR-2′ andPR-2″

About 300,000 plaques of the cDNA library prepared as described from RNAisolated from TMV infected tobacco leaves are screened with a probecomprising a mixture of labelled oligonucleotides (JR138) of theformula:

5′ATGTTYGAYGARAAYAA 3′ (SEQ ID No. 68),

wherein each Y is independently selected from a pyrimidine C or T andeach R is independently selected from a purine G or C. This probe is 17bases long with 16 fold redundancy in the mixture. The probe design isbased on an analysis of the protein data in Example 16. The probe willrecognize clones containing the PR-2, PR-N or basic β-1,3-glucanase butnot PR-O′ or PR-O.

30 positively hybridizing plaques are isolated and purified and theirinserts are subcloned into the bluescript plasmid. The sequence of theinserts is determined by DNA sequencing and the results indicate that atleast three distinct cDNA's have been isolated. When comparing to theprotein data in Example 16, it is clear that one type of clone containsa full-length cDNA that encodes the PR-2 protein (PBSGL117, ATCC 40691),one type encodes the PR-O protein (pBSGL134, ATCC 40690), one typeencodes the PR-N protein (pBSGL125, ATCC 40692; pBSGL148, ATCC 40689)and one type encodes a highly related protein which is neither PR-2,PR-N or PR-O. This type of cDNA is named PR-2′(pBSGL135, ATCC 40685).

In order to isolate a cDNA clone encoding PR-O and also to isolate morefull-length cDNA clones, the library is screened for a second time witha PstI restriction fragment from pBSGL125. pBSGL125 is a 600 base pairclone encoding a PR-N, which is truncated at the 5′ end. About 300,000plaques of the library are screened and 17 positive plaques areisolated, purified and the inserts are subcloned into bluescript. Thesequence of the inserts are determined by DNA sequencing.

To insure that full-length clones are isolated from all of the acidicglucanases, two final strategies are employed. First, a final round ofscreening is carried out using a 210 base pair, PvuII-TaqI, restrictionfragment derived from pBSGLI 17 as a probe. In this screen, 17 clonesare isolated, purified, subcloned into bluescript and their sequence isdetermined by DNA sequencing.

The second strategy is to amplify the 5′ end of the cDNA out of thelibrary by a PCR strategy described in Example 46 above. In this case,the two oligonucleotides B and C are used along with a thirdoligonucleotide JR209 which is complementary to positions 152 to 169 ofthe PR-2 cDNA sequence. In this experiment, two PCR reactions arecarried out; one containing an aliquot of the cDNA library as a templateand the primers JR209 and DP01 (oligo B from Example 46) and the otherusing an aliquot of the cDNA library as a template and using JR209 andGP38 (Oligo C from Example 46) as primers. The sequence of JR209 is asfollows:

JR209 5′AACATCTTGGTCTGATGG 3′ (SEQ ID No. 69).

A positive control in the amplification experiment is an aliquot of thepurified GL117 clone encoding the full-length PR-2 cDNA amplified withJR209 and DP01. A negative control in the experiment is an amplificationusing JR209 and GP38.

Aliquots of the PCR reactions are analyzed by agarose gel elctrophoresisand it is found that a band about the size of the positive controlamplified from the library for each set of primers. This result suggeststhat the procedure is successful and so the remaining DNA is purifiedand then treated with the Klenow fragment of DNA polymerase I in thepresence of all four dNTP's, as described by Maniatis, et al, to makethe ends of the DNA molecules “flush”. The Klenow enzyme is inactivatedby heating to 65° C. for 15 minutes and the DNA is then restricted withEcoRI.

The DNA is purified and then electrophoresed on a 1.5% LGT agarose gel.The band of DNA of the correct size is excised from the gel and used toligate into the bluescript plasmid, which has been restricted with bothEcoRI and EcoRV. The ligation is used to transform bacteria and positivecolonies are selected and analyzed by DNA sequencing.

The result of the preceding procedures is the isolation of clonescomprising the full-length cDNAs for PR-2, PR-N, PR-O, a fourth type ofglucanase designated PR-2′ which encodes an unknown protein, and a fifthtype of glucanase designated PR-2″ which encodes an unknown protein.

pBSGL117 is the isolated plasmid containing a cDNA insert encoding aPR-2 protein. The sequence of PR-2 is included as SEQ ID No. 21.

pBSGL134 is the isolated plasmid containing a cDNA insert encoding aPR-O protein. The sequence of PR-O is included as SEQ ID No. 23.

pBSGL167 is the isolated plasmid containing a cDNA insert encoding aPR-N protein. The sequence of PR-N is included as SEQ ID No. 24.

The phage lambda tobcDNAGL161 is a clone containing the full-lengthPR-2′ cDNA. The sequence of PR-2′ is included as SEQ ID No. 25.

The phage lambda tobcDNAGL153 is a clone containing the full-lengthPR-2″ cDNA. The sequence of PR-2″ is included as SEQ ID No. 26.

The determination of the protein encoded in the cDNA insert is based ona comparison between the protein data in Example 16 and the deducedprotein sequence encoded by the cDNA insert.

pBSGL117, pBSGL125, pBSGL148, pBSGL134 and pBSGL135 have all beendeposited in the American Type Culture Collection, Rockville, Md. asnoted in the list of deposits provided above in section N of theDETAILED DESCRIPTION.

Example 49 Isolation of cDNA Clones Encoding PR4

Approximately 300,000 plaques of the cDNA library described in Example41 are screened with a probe comprising a mixture of labelledoligonucleotides (LF30) of the formula:

5′GGYTTRTCXGCRTCCCA 3′ (SEQ ID No. 70)

wherein each Y is independently selected from a pyrimidine C or T, eachR is independently selected from a purine G or A, and each X isindependently selected from a purine or pyrimidine A, C, G, or T. Thisprobe is 17 bases long with 32 fold redundancy in the mixture. The probedesign is based on an analysis of the protein data in Example 17.

Positively hybridizing plaques are purified and the DNA sequences ofseven inserts determined. Three of the isolates had the same DNAsequence which was called PR-4a.

The sequence of PR-4a is presented as SEQ ID No. 31 and the plasmidpBSPR-4a that contains the insert shown in the Sequence was deposited inthe ATCC (accession no. 75016).

The sequence of PR-4b is presented as SEQ ID No. 32 and the plasmidpBSPR-4b that contains the sequence was deposited in the ATCC (accessionno. 75015).

PR-4a and PR-4b are structurally similar, they may be collectivelyreferred to as “PR-4 proteins.” Other cDNA sequences which hybridizewith either of the aforementioned PR-4 encoding cDNAs have been found tobe hybridize with the other, and are said to encode a polypeptide havingPR-4 activity. See, e.g. Examples 51-58, below.

Example 50 Isolation of a Distinct Family of cDNA Clones Encoding SAR8.2Proteins

The cDNAs designated SAR8.2a, SAR8.2b, SAR8.2c, SAR8.2d, and SAR8.2eshare no significant homology with any known group published DNAsequence, and are therefore considered a group of sequences distinctfrom cDNA sequences that encode other plant pathogenesis-related relatedproteins. Amongst themselves, they share DNA sequence identity rangingfrom 70 to 91% and are therefore considered to be members of a singledistinct family of cDNA clones, called the SAR8.2 encoding DNAsequences. See FIG. 39. Their predicted protein products containputative signal peptides. Assuming cleavage of the signal peptides in afashion consistent with the rules of von Heijne, Nuc. Acids. Res. 14:4683-4690 (1986), the mature proteins encoded by SAR8.2a-SAR8.2d havemolecular weights of 7500-7700. SAR8.2e, due to a duplication of 25amino acids at its carboxy terminus compared to SAR8.2a-SAR8.2d, has apredicted mature molecular weight of 9655. All of the predicted proteinshave calculated isoelectric points greater than or equal to 10.

Transgenic tobacco plants expressing an SAR8.2 cDNA are demonstrablyresistant to the fungal pathogen Phytophthora parasitica, the causativeagent of black shank disease (see Example 165. Thus, an SAR8.2 proteinis one encoded by a DNA sequence that hybridizes to any of the disclosedSAR8.2 cDNA sequences under low stringency conditions (as laid out inExample 46), and which is capable of conferring resistance to fungaldiseases in plants in which it has been expressed.

Filter lifts of the sub-cloned library prepared above are screened withlabeled cDNA probes in a method described previously (St. John and Davis(1979) Cell, 16:443-452 (1979)) except that the same filter lifts arescreened sequentially rather than by screening replicate lifts. Thefirst probe is synthesized asdescribed, using reverse transcriptase and[³2P]-dCTP, from mRNA of the mock-induced RNA sample isolated above;following probing and exposure to X-ray film the same filters are probedagain (without stripping) using probe synthesized as above but from theTMV-induced RNA sample isolated above.

Following exposure the two X-ray films are compared using a computerizeddigital image analysis system (Biological Vision, Inc., San Jose,Calif.) according to the manufacturer's specifications, and plaques areselected that yield an increased signal when probed with the TMV-inducedprobe.

The selected plaques are purified by a second round of probing at aselected plaque density of approximately 100/plate, and the cDNA insertsare recovered from the phage as follows: a small amount of isolatedphage DNA is digested with EcoRI followed by inactivation of therestriction enzyme, dilution, re-ligation with T4 DNA ligase, andtransformation into E. coli strain DH-5, (Bethesda ResearchLaboratories, Bethesda, Md.) followed by selective growth onampicillin-containing culture plates.

This procedure allows direct recovery of the cDNA contained in theplasmid vector from the phage vector. The DNA sequence of a clone thusisolated, designated SAR8.2a, is shown as SEQ ID No. 15. Further clonesthat cross-hybridize with SAR8.2a are isolated by using SAR8.2a as aprobe and re-screening the library under high or low stringencyconditions. Appropriate conditions are those described in Example 46.

A total of 24 phage plaques are identified by this method and their DNAsequences are determined. These 24 clones are found to fall into fiveclasses based on DNA sequence identity: 5 of the 24 clones aredesignated type SAR8.2a, 8 are type SAR8.2b, 4 are type SAR8.2c, 6 aretype SAR8.2d, and one is type SAR8.2e. The sequences of longest memberof each class of clone, appear as SEQ ID Nos. 15-19.

SAR8.2a, b, c, and d are greater than 91% identical to each other in DNAsequence in their open reading frames. As shown in FIG. 40, thepredicted protein products from these same clones are between 88 and 96%identical to each other in amino acid sequence. SAR8.2e is 71% identicalto SAR8.2d in DNA sequence, and 72% identical in amino acid sequence.

Hybridization of tobacco SAR8.2 cDNAs to genomic DNA from other plantsis detected by genomic Southern blot analysis (see Example 4) under lowstringency hybridization and washing conditions. Appropriate conditionsare hybridization at 42° C. in 30% formamide, 5×SSC 0.1% SDS, 5 mM EDTA,10×Denhardt's solution, 25 mM sodium phosphate pH 6.5, and 250 g/mlsheared salmon sperm DNA; washing at 42° C. in 2×SSC, 0.1% SDS (where1×SSC is 150 mM NaCl, 15 mM Na citrate). Alternatively, the lowstringency conditions described in Example 46 can be used. Positivehybridization to multiple discrete bands is detected in genomic DNA fromseveral Solanaceae, including Lycopersicon esculentum, as well asBrassica, Ricinus, and Arabidopsis.

SAR8.2-encoding DNA sequences can be isolated from other plants by usingthe tobacco SAR8.2a, b, c, d or e cDNA sequence as a probe for screeningcDNA or genomic libraries of the plant of interest under low stringencyconditions, as described in the present Example or in Example 46.

Analysis of RNA from mock-induced vs TMV-induced tobacco leaves, usingNorthern analysis and Primer extension assay confirms that steady-statelevels of the pSAR8.2 family mRNA's are increased by TMV induction.

Example 51 Isolation of cDNA Clones Encoding the Chitinase/Lysozyme FromCucumber

Two regions of the protein sequence determined in Example 18, above, areselected and oligonucleotide probes are synthesized that arecomplementary to all possible combinations of messenger RNA capable ofencoding the peptides. The sequences of the probes are:

Probe 1:    G T    A 5′-CCATTCTGNCCCCAGTA-3′ (SEQ ID No. 71) Probe 2:   G G G G C 5′-GGATTATTATAAAATTGNACCCA-3′. (SEQ ID No. 72)

About 300,000 plaques are plated from the library constructed above andduplicate plaque lifts are probed either with ³²P labeledoligonucleotide mixture 1 (probe 1) or mixture 2 (probe 2). Plaques areisolated that show positive results when screened with either probe.Isolation of phage and automatic excision are carried out as describedin the Stratagene Lambda Zap laboratory manual.

Once the chitinase cDNA clones are isolated in the bluescript plasmidthey are sequenced by dideoxy sequencing. The sequence of the chitinasecDNA contained in the plasmid pBScucchi/chitnase is presented in SEQ IDNo. 3.

Example 52 Isolation of cDNA Clones Encoding Chitinase/Lysozymes FromTobacco

About 300,000 plaques of the TMV-infected tobacco cDNA library describedin Example 41 are screened using a labeled cDNA probe encoding thecucumber chitinase/lysozyme cDNA and washing filters at 50° C. in 0.125mM NaCl, 1% SDS, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA. Positiveplaques are purified and the DNA sequence of two clones, named pBSCL2and pBSTCL226 are determined. These are presented in SEQ ID Nos. 29 and30, respectively. The proteins encoded in the clones of these sequencesare determined to be chitinase/lysozymes based on structural homology tothe cucumber chitinase/lysozyme.

In addition, a protein is purified from intercellular fluid ofTMV-infected tobacco as described in Example 16. Peptides are generatedand sequenced as described in Example 9.

The protein encoded by pBSTCL226, corresponding to an acidic isoform ofchitinase/lysozyme, was found to match the deduced peptide sequencesexactly.

Example 53 Cucumber Peroxidase cDNA

An oligonucleotide probe is designed to isolate cDNA's encoding thecucumber peroxidase based on the protein data presented in Example 50.The sequence of the mixture of oligonucleotides is as follows:

JR74 5′ACRAARCARTCRTGRAARTG 3′ (SEQ ID No. 73),

wherein each R is independently selected from a purine G or C. Thisoligonucleotide mixture is 20 bases long and contains 64 species.

A cDNA library is prepared from RNA isolated from leaves of cucumberplants five days after infection with tobacco necrosis virus asdescribed in Example 43. This library is constructed in the Lambda ZAPcloning vector.

About 300,000 plaques of this cDNA library are screened with theoligonuleotide probe and 25 plaques are isolated. These are rescreenedseveral times and purified. As a result of this process only six plaquesremained as still positive after many rounds of purification. Theinserts contained in these clones are excised using the automaticexcision protocol described in the Stratagene Lambda ZAP laboratorymanual. The inserts are sequenced by double stranded dideoxy sequencingand then the structures are analyzed by dot matrix analysis comparing tothe sequence of an acidic, lignin-forming peroxidase isolated fromtobacco (Lagrimini, et al., Proc. Natl. Acad. Sci. USA 84: 7542-7546(1987)).

Two of the clones, Perl and Per25, show some limited homology to thetobacco cDNA and are chosen for further analysis. Upon completesequencing of the clones, it is found that they encode the same protein.

A probe derived from the cucumber peroxidase is then used to rescreen tocucumber library and about 30 plaques are isolated. After purificationthe inserts are excised from the phage as plasmids and then the sequenceis determined by DNA sequencing. The results of this analysis is theisolation of two types of peroxidase cDNA clones from cucumber. Oneencoding a basic protein encoded by the plasmid pBSPERI, the nucleotidesequence which is shown in SEQ ID No. 22, and the other encoding arelated peroxidase contained in the plasmids, pBSPER24 and pBSPER25.

Plasmids pBSPERI, pBSPER24 and pBSPER25 have all been deposited in theAmerican Type Culture Collection as noted in the list of depositsprovided above in section N of the DETAILED DESCRIPTION.

As disclosed above, also included within the scope of the presentinvention are cDNA sequences that hybridize with the enumeratedSequences and encode a polypeptide having the activity of the plant PRprotein encoded by the enumerated Sequence with which it hybridizes. Therepresentative Examples set forth below describe a protocol sufficientlydetailed to guide or instruct one of ordinary skill in the art toisolate other cDNA's encoding plant PR proteins within the scope of theinvention without undue experimentation.

Example 54 Construction of a Chemically-Induced Arabidopsis cDNA Library

Mature Arabidopsis thaliana ecotype Columbia (Lehle Seeds, Tucson,Ariz.) plants are sprayed with a 0.5 mg/ml suspension of a wettablepowder formulation of methylbenzo-1,2,3-thiadiazole-7-carboxylateconsisting of 25% active ingredient. Seven days later, the leaf tissueis harvested and frozen in liquid N₂. Total RNA is isolated as describedin Example 6 and Poly (A)⁺ RNA is isolated using a Poly (A) Quik™ mRNAisolation kit from Stratagene (La Jolla, Calif.). This Poly (A)⁺ RNA isthen used to make a cDNA library in the uni-zap™ XR vector (Stratagene)using a ZAP-cDNA™ Gigapack® II Gold cloning kit from Stratagene. Aportion of the cDNA library is amplified as described in the Stratagenekit.

Example 55 Isolation of an Acidic beta-1,3-glucanase EDNA FromArabidopsis

Arabidopsis thaliana ecotype Columbia plants are sprayed with a 1 mg/mlsuspension of a wettable powder formulation of 2,6-dichloroisonicotinicacid methyl ester, consisting of 25% active ingredient. After sevendays, the leaves are harvested and the intercellular fluid (ICF)collected as described in Example 12.

The proteins contained in the ICF are fractionated on a 20%polyacrylamide native gel (Phast system, Pharmacia). Two predominantchemically-induced protein bands are detected. The slower migrating(top) band is designated band 1. The faster migrating band (band 2) isexcised from the gel and the protein eluted. Peptides are generated withlysyl endopeptidase and sequenced as described in Example 9.

The deduced peptide sequences were found to align with a high degree ofsimilarity with the sequence of several known beta-1,3-glucanases fromtobacco. An oligonucleotide of sequence:

5′ATG TTY GAY GAR AAY AA 3′ (SEQ ID No. 74)

that corresponds to the amino acids MFDENN (SEQ ID No. 75), which arehighly conserved among acidic, extracellular beta-1,3-glucanases (Wardet al., Plant Physiol. 96: 390-397 (1991), is used as a hybridizationprobe to isolate cDNA clones from the A. thaliana library described inExample 54. The clones are purified, plasmids excised in vivo asdescribed by the manufacturer of the cloning vector (Stratagene), andtheir nucleotide sequences determined. The partial DNA sequence of thelongest of these was cloned into pAGL2, (ATCC No. 75048).

Example 56 Isolation of a PR-1-Related cDNA Clone From Arabidopsis

Approximately 200,000 plaques from the Arabidopsis cDNA library arescreened with a combination of PR-1a and PR-1 basic cDNAs from tobaccoas probes at low stringency, as described in Example 46.

Three purified positive clones are in vivo excised (as per Stratagene)and plasmid DNA is isolated and sequenced. All 3 cDNAs share sequenceidentity and one cDNA clone appears to be full length (clone pAPR1C-1,ATCC No. 75049) as determined by comparison to the tobacco PR-1sequences. The DNA sequence appears as SEQ ID No. 33.

Proteins from the ICF described in Example 55 are separated by SDSpolyacrylamide gel electrophoresis on an 8-25% acrylamide gradient gel(Phast system, Pharmacia). A third abundant chemically-induced proteinis identified that migrates at an apparent molecular weight similar tothat of the PR-1 proteins of tobacco. The protein band is excised fromthe gel, and is separated from a closely migrating protein by reversephase HPLC. Peptides from the chemically-induced protein are generatedwith trypsin, and their sequences determined as described in example 9.

Peptides are also generated with lysyl endopeptidase as described inExample 9. The sequence of one peptide is determined as described inExample 9.

The predicted protein encoded by the C-1 clone was found to match thededuced peptide sequences exactly, indicating that the C-1 clone encodesthe extracellular Arabidopsis protein related to PR-1 from tobacco.

Example 57 Isolation of a PR4-Related cDNA Clone From Arabidopsis

Approximately 200,000 plaques from the Arabidopsis cDNA library areprobed with the tobacco PR-4 cDNA clone at low stringency as describedin Example 46. Four purified positive clones are in vivo excised and theresultant plasmid inserts are sequenced. The DNA sequence of one ofthese clones, designated pSLP1 (ATCC #75047) appears as SEQ ID No. 34.The clone is found to have substantial sequence homology to the win1 andwin2 genes of potato (Sanford et al, Mol. Gen. Genet. 215: 200-208(1988)). Specifically, it contains the “hevein” domain at its predictedN-terminus, which is also found in the tobacco basic chitinase (Shinshiet al., Plant Mol. Biol. 14:357-368 (1990)). The clone has homology toPR-4 from tobacco at the predicted C-terminus.

Example 58 Isolation of a PR-5-Related cDNA Clone From Arabidopsis

The band 1 protein described in Example 55 is eluted from a nativepolyacrylamide gel and peptide generated and their sequences determinedas described in example 9.

The peptide sequences from the band 1 protein were found to have asignificant level of similarity to osmotin and PR-R (a.k.a PR-5) oftobacco. Using these peptide sequences, oligos SU01, SU02, and EW59 weredesigned. SU01 is a 128-fold degenerate oligonucleotide mixture 17 basesin length having sequence:

5′AAY AAY TGY CCN ACN AC 3′ (SEQ ID No. 76)

(where Y=C or T; N=C or T or A or G)

which is derived by reverse translation from the amino acid sequenceNNCPTT SEQ ID No. 77), which occurs in the N-terminus of the protein.SU02 is a 32-fold degenerate oligonucleotide mixture of sequence:

5′AC RTC RTA RAA RTC YTT 3′ (SEQ ID No. 78)

(where R=A or G)

which is the antisense strand of a sequence derived by reversetranslation from the amino acid sequence KDFYDV (SEQ ID No. 79).

EW59 is a 64-fold degenerate oligonucleotide mixture 17 bases in lengthhaving the sequence:

5′CK RCA RCA RTA YTG RTC 3′ (SEQ ID No. 80)

(where K=T or G)

which is the antisense strand of a sequence derived by reversetranslation from the amino acid sequence DQYCCR (SEQ ID No. 81).

A polymerase chain reaction is carried out using double-stranded cDNA(used for constructing the library described in Example 54) as template,and SU01 and EW59 as primers. The 50 ul reaction contains approximately20 ng cDNA, 12 uM SU01, 6 mM EW59, 200 uM each dAPT, dCTP, dGTP, anddTTP, 1×PCR buffer (Perkin-Elmer Cetus), and 2.5 u Amplitaq polymerase.The reaction is cycled 30 times through a temperature profile of 94° C.for 30 sec., 44° C. for 45 sec., and 72° C. for 30 sec in a DNA thermalcycler (Perkin Elmer Cetus, Norwalk, Conn.).

A band approximately 450 bp in length is amplified that also hybridizesto SU02, which is predicted to lie between SU01 and EW59, based onalignment of the peptides with the osmotin and PR-R sequences. Thefragment is gel purified, labeled by random printing, and used to probethe cDNA libray. Four positively-hybridizing clones are purified, invivo excised, and their DNA sequences determined.

The sequence of one clone, designated pATL12a (ATCC #75050), appears asSEQ ID No. 35. This clone appears to be a full-length clone based onidentification of the N-terminal peptide sequence in the predictedprotein coding sequence near the 5′ end of the clone.

Example 58A Cloning of Class IV Chitinase cDNAs From Arabidopsis

Class specific degenerate oligonucleotides were designed from areas ofhomology between bean PR4 chitinase (Margis-Pinheiro et al., Plant Mol.Biol. 17: 243-253 (1991)) and sugar beet chitinase 4 (Mikkelsen et al.,in “Advances in Chitin and Chitosan, ed by Brine et al., pub. byElsevier, Amsterdam (1992)): oligonucleotides were designed degeneratefor the peptide sequences HFCYIEE (forward spanning nucleotides406-426), and IRAING (reverse, spanning nucleotides 705 to 675). DNA wasextracted from two-week old plants of Arabidopsis thaliana ecotypeLandsberg and amplified using a Perkin-Elmer thermal cycler 480 at thefollowing cycle settings: 94 C for 5 minutes; 35 cycles at 94 C, 1minute, 43 C or 45 C, 1 minute, and 72 C, 2 minutes; followed by 5minutes at 72 C. The amplified fragment was gel purified, collected bycentrifugation through Whatman paper, ethanol precipitated, resuspendedin TE, digested with BamHI and NsiI and subcloned into pTZ18U(Pharmacia). Four clones of the fragment were sequenced; they differedonly within the oligo-derived sequence as could be expected fromamplification with degenerate oligonucleotides.

Two different cDNAs were isolated simultaneously by screening a leaftissue cDNA library (Uknes et al., Plant Cell 4: 645-656 (1992)) at highstringency with the PCR amplified genomic fragment described above.Duplicate plaque lifts were taken with nitrocellulose filters(Schlcicher & Schuell, Keene, N.H.) (Ausubel et al., in “CurrentProtocols in Molecular Biology, pub. by J. Wiley & Sons, New York(1987)). Probes were labelled by random priming (using the labelingsystem supplied by Gibco BRL, Gaithersburg, Md.). Hybridization andwashing were done at 65 C according to Church and Gilbert, Proc. Natl.Acad. Sci. 81: 1991-1995 (1984). Positive plaques were purified andplasmids containing the cDNA inserts were in vivo excised for DNAsequence determination. Of the six positive clones two contained aninsert with structural homology to previously characterized class IVchitinases and were designated class IV chitinase type A and four cloneswere divergent in that they lacked the class IV chitinase hevein domain;these were designated class IV chitinase type B. As none of the cDNAinserts was full-length, an additional 29 bp of class IV chitinase typeA and 17 bp of class IV chitinase type B, both containing a methionineintiation codon were amplified from ethephon-induced RNA using the 5′RACE system for rapid amplification of cDNA ends (Gibco BRL,Gaithersburg, Md.). Sequence comparisons were performed using the GAPand PILEUP features of the Genetics Computer Group software (GeneticsCompute Group, Madison, Wis.).

Class IV chidnase type A is a 1079 base pair cDNA with an open readingframe of 264 amino acids containing the characteristic cysteine-richhevein and chitinolytic domains and the three short deletions typicallyfound in class IV chitinases (see SEQ ID No. 37). The cDNA for class IVchitinase type B is 952 base pairs in length and encodes a protein of214 amino acids which lacks a hevein domain and contains a fourthdeletion (see SEQ ID No. 38). The cDNAs are 71% identical overall and80% identical over coding sequence. The predicted protein encoded byArabidopsis class IV chitinase type A is 89% homologous to the basicBrassica napus (rapeseed) class IV chitinase, 61% homologous to basicBeta vulgaris (sugar beet) class IV chitinase, 57% homologous to basicZea mays class IV chitinase B, 58% homologous to acidic Phaseolisvulgaris (bean) PR4 class IV chitinase, and 55% homologous to acidicDioscorea japonica (yam) class IV chitinase. It is 42% homologous toArabidopsis thaliana basic class I chitinase.

The predicted mature protein encoded by Arabidopsis class IV chitinasetype A has a molecular weight of 25695 D and a pI of 7.8; whereas theprotein encoded by Arabidopsis class IV chitinase type B has a molecularweight of 20553 D and a pI of 10 assuming the removal of a signalpeptide based on homology to tobacco PR-Q.

Northern analysis showed that both chitinases were induced by TCVinfection, confirming their classification as PR-proteins.

Using techniques well known in the art, these cDNAs can be cloned intoexpression cassettes and vectors for transfer to trarsgenic plants.Typical techniques used in the art are described in section 6 (examples64 to 82), section 7 (examples 83 to 108) and section 8 (examples 109 to133).

Example 58B Isolation of Wheat cDNAs Specifically Induced by Treatmentwith benzo-1,2,3-thiodiazole-7-carboxylic acid thiomethyl ester, Usingthe Method of Differential Plaque Filter Hybridization

Samples of winter wheat (cultivar Kanzler) were harvested 2-3 days aftertreatment with either water or 200 ppm of the plant activator compoundbenzo-1,2,3-thiodiazolecarboxylic acid. Total RNA was prepared fromfrozen tissue samples using a standard phenol extraction/LiClprecipition procedure (Lagrimni et al., Proc. Natl. Acad. Sci. 84:7542-7546 (1987)). PolyA (+) RNA was purified from total RNA using thePoly(A) Quik mRNA purification kit (Stratagene Cloning Systems, LaJolla,Calif.). A bacteriophage lambda ZAP II cDNA library was prepared fromthe benzo-1,2,3-thiodiazolecarboxylic acid treated polyA(+) sample usingthe Uni-Zap XR Gigapack II Gold cloning kit (Stratagene) as described bythe manufacturer. The phage library was plated at a density ofapproximately 5000 plaques on a 10 cm petri dish and grown for 6-8 hoursat 37 C. Duplicate filter lifts of the plaques were made usingnitrocellulose membranes (Schleicher & Schuell, Keene, N.H.). Labelledfirst strand cDNA probes were prepared from polyA of both the watercontrol and the benzo-1,2,3-thiodiazlecarboxylic acid-treated samplesusing 32P-dCTP and the AMV reverse transcriptase (GibcoBRL,Gaithersburg, Md.) under the manufacturer's conditions. Each probe washybridized (>106 cpm/ml) with one set of the duplicate lifts overnightat 65 deg C. Hybridization and wash conditions were as described inChurch and Gilbert, Proc. Natl. Acad. Sci. 81: 1991-1995 (1984).Hybridization was detected by autoradiography.

Plaques appearing to hybridize preferentially to the chemically treatedcDNA were purified and their cDNA inserts were amplified using theGeneAmp Polymerase Chain Reaction (PCR) kit (Perin Elmer, Norwalk,Conn.) and primers homologous to the flanking lambda Zap II sequences.The amplified inserts were excised from a low melting temperatureSeaPlaque GTG agarose gel (FMC BioProducts, Rockland, Me.) and labelledusing 32P-dCTP to and the Random Primers DNA Labeling System (GibcoBRL). These probes were hybridized with total RNA blots (Ausubel et al.,in “Current Protocols in Molecular Biology, pub. by J. Wiley & Sons, NewYork (1987)) of control and benzo-1,2,3-thiodiazolecarboxylic acidtreated RNAs to verify that they contained chemically induced cDNAs.

The induced clones were in vivo excised into pBluescript plasmidsaccording to the manufacturer's instructions (Stratagene) and plasmidDNAs were purified using Magic Miniprep columns (Promega Biotech,Madison, Wis.). The cDNA sequences were determined by the chaintermination method using dideoxy terminator labelled with fluorescentdyes (Applied Biosystems, Inc., Goster City, Calif.). The DNA and thepredicted amino acid sequences were compared to available databasesusing the GAP (Deveraux et al., Nucl. Acids Res. 12: 387-395 (1984)) andthe BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)) programs.

The nucleotide sequence of one apparently full length induced clone isset forth in SEQ ID No. 39. This clone, denoted WCI-1 (Wheat ChemicalInduction), was found to share limited homology with two rice cDNAs, oneexpressed specifically in the shoot apical meristem (De Pater andSchilperoort, Plant Mol. Biol. 18: 161-164 (1992)), the other inducibleby salt stress (Claes et al., Plant Cell 2: 19-27 (1990)). The functionof these proteins is unknown.

The predicted amino acid sequence of a second highly induced cDNA,WCI-2, clearly identified it as an isozyme of wheat lipoxygenase, basedon its homology to other plant lipoxygenases in the database. The DNAsequence of this cDNA is shown in SEQ ID No. 40.

A third class of induced cDNA (WCI-3) was isolated which to date showsno significant homology to sequences in the databases. The DNA sequenceof this apparently full-length clone is set forth in SEQ ID No. 42.

Using techniques well known in the art, these cDNAs can be cloned intoexpression cassettes and vectors for transfer to transgenic plants.Typical techniques used in the art are described in section 6 (examples64 to 82), section 7 (examples 83 to 108) and section 8 (examples 109 to133).

Example 58C Isolation of Wheat cDNAs Specifically Induced by TreatmentWith benzo-1,2,3-thiodiazolecarboxylic acid, Using the Method ofDifferential cDNA Display

The total and polyA(+) samples described in Example 1 were used for PCRdifferential display of the mRNA from water andbenzo-1,2,3-thiodiazolecarboxylic acid treated wheat tissue essentiallyas described in Liang and Pardee, Science 257: 967-971 (1992). AmplifiedcDNA fragments that appeared to be present only in the chemicallytreated sample were excised from the dried sequencing gel andelectroeluted using a Centrilutor device and Centricon-30Microconcentrators (Amicon, Beverly, Mass.). The purified fragments werePCR amplified using primers that consisted of the original differentialdisplay 10-mers plus an additional 10 bases of unique sequence added totheir 5′ ends. After 8 PCR cycles at low annealing temperature (42-45C), the annealing temperature was raised to 60 C for an additional 30cycles. Essentially 100% of the fragments could then be visualized byEtBr staining and were excised from a SeaPlaque GTG agarose gel (FMC).

These gel fragments were labelled and used to probe RNA blots aspreviously described. Fragments that hybridized only with chemicallytreated RNA were TA-cloned into the plasmid vector pCR II using a TACloning Kit (Invitrogen Corporation, San Diego, a Calif.). Inserts fromthe plasmids were screened again against control/chemical RNA blots toverify that the inducible gene fragment had been subcloned. Inducedfragments were then labelled by random priming and hybridized againstfilter lifts of the chemically induced cDNA library as described inExample 58A. Hybridizing plaques were purified, sequenced, and analyzedas previously described in order to obtain full length clonescorresponding to the original small (200-400 bp) fragments, and toidentify the induced gene product where possible.

The nucleotide sequence of one clone obtained by this procedure is setforth in SEQ ID No. 43. This clone, WCI-4, has some homology to knownthiol protease sequences from a variety of sources and may therefore bea thiol protease.

Partial sequences of an additional induced gene that was isolated usingdifferential display is set forth in SEQ ID No. 44. This fragment showsno database homology and has been designated WCI-5.

Using techniques well known in the art, these cDNAs can be cloned intoexpression cassettes and vectors for transfer to tansgenic plants.Typical techniques used in the art are described in section 6 (examples64 to 82), scion 7 (examples 83 to 108) and section 8 (examples 109 to133).

Example 59 Isolation of the cDNA Encoding the Acidic Form ofβ-1,3-glucanase

About 300,000 plaques of the tobacco cDNA library described above arescreened using a labeled cDNA probe encoding the basic form ofβ-1,3-glucanase (Shinshi, H. et al., supra) and washing filters at 50°C. in 0.125 M NaCl, 1% SDS, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA.15 positive plaques are isolated and the insert is subcloned into thebluescript plasmid. Partial DNA sequencing reveals that clone 5 and 6encode identical cDNA's which have about 55% homology to the known DNAsequence of the basic form of β-1,3 glucanase. The sequence of the cDNAin clone 5 and 6 is determined and shown in SEQ ID No. 8. It isconcluded that this cDNA encodes the acidic form of β-1,3 glucanasebased on the limited amino acid sequence homology and the more acidicisoelectric point of the encoded protein.

3. A Novel, Differential Cloning and Screening Technology

Example 60 Differential Enrichment Scheme

The following is a specific example of the differential cloning andscreening technique described above in section L of the “DETAILEDDESCRIPTION” using cDNA populations in phage cloning vectors. The“induced” and “uninduced” cDNA populations (constructed from mRNA fromTMV-induced or mock-induced tobacco leaves in this case) are cloned intwo different phages so that the primers used for amplifying the cloneinserts will be different (and thus not hybridize in later steps).

The “induced” cDNA bank is constructed in GEM-4 (Promega, Inc.) and the“uninduced” or “mock-induced” DNA bank is constructed in ZAP-II(Strategene, Inc.). For each vector specific oligonucleotide primers aresynthesized such that they represent sequences in the phage vectorimmediately adjacent to the cDNA cloning site, and that, when used inthe polymerase chain reaction in the presence of the correspondingphage, the DNA cloned into the cDNA cloning site will be amplified.

A population of phages representing all the members of each bank areamplified together, producing a population of cDNA inserts representingthe bank. One of the oligonucleotide primers in each case isbiotinylated so that a specific strand of each amplified bank DNA can bepositively selected by avidin affinity chromatography and strandseparation, followed by release and recovery of the biotin-selectedstrand.

Importantly, the primers used for biotinylation are selected such thatstrands of opposite polarity are selected from the “induced” vs“uninduced” cDNA banks. The biotin tag in the case of the “induced” DNAis a labile one in that the spacer are through which the biotin moietyis attached contains a disulfide linkage, and the tag in the “uninduced”DNA is a stable one. In this case the single strand from the “induced”population is released by dithiotreitol treatment, losing its biotintag, and the single strand from the “uninduced” population is releasedby denaturation of the avidin molecule, while retaining its biotin tag.

When the recovered “target” DNA (single strand from the “induced”library) is hybridized to the recovered “driver” DNA (single strand fromthe “mock-induced” or “uninduced” library), the complexes that areformed, and the excess “driver” DNA can be removed by avidin affinitychromatography.

The remaining “target” DNA still bears the primer sequences, making itsrecovery, by subsequent repair or amplification and cloning, verysimple.

In the alternate scheme also described above in section L of the“DETAILED DESCRIPTION”, both the “target” and “driver” single strandedDNAs are recovered by denaturation of the avidin on the affinity matrix,and both retain their biotin tags. After hybridization all the moleculesare bound to the affinity matrix and following washing, thenon-hybridized “target” DNA, bearing the “liable” affinity tag, isselectively eluted from the matrix by dithiothreitol. This modificationallows a positive selection for the single stranded “target” DNA,avoiding potential problems with less-than-quantitative binding of thehybridization mix to the affinity matrix.

The advantage of this technique over those previously described is theability to isolate those genes which are turned on only to low levels,in specific circumstances, and which may play a causative role in someimportant biological phenomenon.

In both forms of the technique described above, the “target” and“driver” single stranded DNA is generated by biotin-avidin affinitychromatography.

An alternative method for generating these single-stranded populationsis a method described as “asymmetric” PCR (Gyllensten, U. and Erlich, A,Proc. Natl. Acad. Sci. USA. 85: 7652-7656 (1988)). This consists ofmultiple cycles of polymerase extension and denaturation, as in PCR, butin the presence of only one of the primers. The primer chosen determinesthe polarity of the resultant DNA, thus allowing the selective polaritycritical to the technique as described above. Asymmetric PCR in thiscase is most easily accomplished using as template a small amount ofdouble-stranded PCR products of the relevant population, from which theexcess primers have been removed.

F. ISOLATION OF NOVEL PR PROTEIN GENES

The following examples disclose the isolation and characterization ofnovel genomic clones encoding PR proteins.

Example 61 Isolation of a Genomic Clone Encoding PR-1′

A genomic library is constructed and screened with a PR-1 cDNA probe asdescribed in Example 20. A clone is isolated and designated lambdatobchrPR1019. A preliminary restriction map is established A ClaIfragment is subcloned into bluescript and then a restriction map ofplasmid, pBS-PR1019Cla, is established A large XhoI fragment is deletedfrom pBS-PR1019Cla resulting in the plasmid pBS-PR1019Cla Xho. Thisplasmid contains about 2.7 kb of tobacco DNA containing the PR1019 gene.Deletions are made in this plasmid and the set of deletions are used forDNA sequencing.

The DNA sequence for 2256 bp of this gene is shown in SEQ ID No. 2. Theprotein encoded in the PR1019 (SEQ ID No. 46) is found to be about 65%homologous to PR-1a, PR-1b, or PR-1c; therefore, this new gene is namedPR-1′.

Example 62 Isolation of a Genomic Clone Encoding the Cucumber ChitinaseGene A. Preparation of Cucumber Genomic DNA

Nuclei are isolated from leaves of Cucumis sativus cv. Long Marketer byfirst freezing 35 grams of the tissue in liquid nitrogen and grinding toa fine powder with a mortar and pestle. The powder is added to 250 ml ofgrinding buffer (0.3 M sucrose, 50 mM Tris, pH 8, 5 mM magnesiumchloride, 5 mM sodium bisulfite, 0.5% NP4O) and stirred on ice for 10minutes. This mixture is filtered through six layers of cheesecloth, andthe liquid is centrifuged at 700×g for 10 minutes. The pellets areresuspended in grinding buffer and recentrifuged. The pellets are againresuspended in grinding buffer and this suspension is layered over a 20ml cold sucrose cushion containing 10 mM tris, pH 8, 1.5 mM magnesiumchloride, 140 mM sodium chloride, 24% sucrose, 1% NP4O. These tubes arecentrifuged at 17,000×g for 10 minutes. The pellets at this stagecontain mostly nuclei and starch granules. High molecular weight DNA isisolated from the nuclei essentially according to Maniatis, T. et al.,Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982) usingcesium chloride ethidium bromide gradients.

B. Preparation of Genomic Library

Three ug of the purified DNA is digested with EcoRV, EcoRI, BamHI orHinDIII and the fragments are separated on a 0.5% agarose gel. The gelis blotted to nylon membrane (GeneScreen Plus, NEN Research Products,Boston, Mass.) in 0.4 M NaOH as described by Reed et al., Nucleic AcidsRes. 13: 7207-7221 (1985). The gel blot is hybridized to a probe of thecucumber chitinase cDNA (SEQ ID No. 3) labeled with 32P by randompriming using the PrimeTime kit (International Biotechnologies, NewHaven, Conn.). A single band of about 12 kb is detected in the EcoRIdigest; thus it is decided to isolate the cucumber chitinase gene usinga total EcoRI digest and ligating into a lambda replacement-type cloningvector.

Ten ug of this DNA is digested to completion using EcoRI and the DNA isthen extracted with phenol, precipitated with ethanol and ligated intothe EcoRI site of EMBL 4. About 100,000 plaques are screened with the32P-labeled cDNA probe of cucumber chitinase. Ten positive plaques arepicked and purified and insert DNA is prepared using LambdaSorbImmunoaffinity Adsorbent according to the manufacturer's instructions(Promega, Madison, Wis.). The insert DNA is analyzed by preliminaryrestriction digest mapping which shows that the inserts are identical.One clone is picked for further analysis; the 12 kb insert (SEQ ID NO:36) is subcloned into the pBluescript vector (Stratagene, LaJolla,Calif.) to give the plasmid pBScucchrcht5 (ATCC accession no. 40941).

C. Preparation of Clones for Sequencing

A restriction map is established for pBScucchrcht5 and fragments of thegenomic DNA insert are subcloned into pBluescript. The clones generatedare designated D1-3, D-Cla1, D-Cla2, D-BamCla, and D-XbaI. The D1-3clone is made from a gel-purified EcoRI fragment of pBScucchrcht5. Thisfragment is digested with EcoRV and the EcoRV insert is gel-purifiedaway from the vector piece. Low melting point agarose forgel-purification is from BRL (Gaithersburg, Md.). EcoRI linkers (BRL)are kinased and ligated with the EcoRV piece, and then the piece isdigested with EcoRI and gel-purified again. The purified fragment isligated with pBluescript that has been digested with EcoRI and treatedwith calf intestinal alkaline phosphatase (Boehringer MannheimBiochemicals, Indianapolis, Ind.).

pBScucchrcht5 is digested with ClaI to generate two ClaI fragmentscontaining insert DNA. These pieces are gel-purified and clonedseparately into pBluescript that has been digested with ClaI and treatedwith calf intestinal alkaline phosphatase. The resulting plasmids arecalled D-Cla1 and D-Cla2. The plasmid D-BamCla is made by isolating theBamHI/ClaI fragment of pBScucchrcht5 and cloning it into pBluescriptdigested with BamHI and Clal. The plasmid D-XbaI is made by isolatingthe insert XbaI fragment of pBScucchrcht5 and cloning it intopBluesccipt digested with XbaI and treated with calf intestinal alkalinephosphatase.

The ligation reactions for each of the constructs are melted byincubating at 65° C. for 10 minutes. 10 ul of this solution are added to30 ul of TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA), mixed and allowed tostand at room temperature. The diluted DNA solution is added to 200 ulof thawed frozen competent cells (E. coli strain DH5) and allowed tostand on ice for 20 minutes. The cells are then heat-shocked for 90seconds at 42° C., followed by a 10 minute room temperature incubation.0.8 ml of SOC medium [Hanahan, D., J. Mol. Biol. 166, 557-580 (1983)] isadded and the culture is incubated at 37 C for one hour. 100 ul of theculture is plated on LB plates [Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, New York (1972)] containing 100ug/ml ampicillin (L-amp) and the plates are incubated overnight at 37°C. Positive colonies are picked and restreaked to a second L-amp plateand the plates are incubated overnight at 37° C.

Plasmid DNA for each of the clones is prepared essentially according toManiatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, NewYork (1982). The DNA sequences of the plasmid inserts are determined bythe dideoxy method using the Sequenase kit (united States Biological,Cleveland, Ohio). The −20 and reverse primers for the pBluescript vectorare used in the sequencing reactions as well as internal primerssynthesized by automated phosphoramidite synthesis on an AppliedBiosystems Synthesizer (Foster City, Calif.). The DNA Star (Madison,Wis.) sequence compilation software is used to compile the sequences ofthe subclones. The total EcoRI genomic clone is shown in SEQ ID No. 36.

The compiled sequence revealed the existence of three open readingframes within the total 12 kb genomic clone (pBScucchrcht5). The openreading framed included in the plasmid D-Cla1 potentially encodes for aprotein of about 31,000 Daltons, with a pI=4.69. The sequence found inthe plasmid D-BamCla corresponds to the sequence of the cDNA (SEQ ID No.3) isolated by Metraux et al., PNAS USA 86: 896-900 (1989). This openreading frame codes for a protein of about 28,000 Daltons with a pI ofabout 4.13. The last open reading frame is included in the plasmidD-Cla2 and potentially codes for a protein of about 29,000 Daltons and apI of about 3.88. These three open reading frames share about 90% DNAsequence homology, but their flanging sequences have diverged.

D. Preparation of Constructs Containing Cucumber Chitinase Gene

1. Construction of pCIB2001

TJS75Kan is first created by digestion of pTJS75 [Schmidhauser et al.,J. Bacteriol. 164: 446-455 (1985)] with NarI to excise the tetracyclinegene, followed by insertion of an AccI fragment from pUC4K [Messing etal., Gene 19: 259-268 (1982)] carrying a NptI gene. pCIB200 is then madeby ligating XhoI linkers to the EcoRV fragment of pCIB7 (containing theleft and right T-DNA borders, a plant selectable nos/NptI chimeric geneand the pUC polylinker [Rothstein et al., Gene 53: 153-161 (1987)] andcloning the XhoI-digested fragment into SalI-digested TJS75Kan. pCIB2001is made by cloning a new polylinker into the multiple cloning site ofpCIB200 to give more unique restriction enzyme sites.

2. Construction of pCIB2001/BamChit

The plasmid pCIB2001/BamChit is constructed by digesting the plasmidDBamCla (Section c, above) with BamHI and KpnI and cloning into pCIB2001that has been digested with BglII and KpnI. pCIB2001/BamChit contains2613 bp of the promoter region from the cucumber chitinase genomicclone, the chitinase gene and approximately 2.1 kb of 3′ sequence.

3. Construction of pCIB2001/SalChit

The plasmid pCIB2001/SalChit is constructed by digesting DBamCla withSalI and cloning into pCIB2001 that has been digested with SalI andtreated with calf intestinal alkaline phosphatase. pCIB2001/SalChitcontains 1338 bp of the promoter region from the cucumber chitinasegenomic clone, the chitinase gene and approximately 2.1 kb of 3′sequence.

4. Construction of pCIB2001/NcoChit

The plasmid pCIB2001/NcoChit is constructed by digesting DBamCla withNcoI, ligating with an NcoI/BamHI adapter, then digesting with BamHI andKpnI, and cloning into pCIB2001 that has been digested with BglII andKpnI. pCIB2001/NcoChit contains 222 bp of the promoter region from thecucumber chitinase genomic clone, the chitinase gene and approximately2.1 kb of 3′ sequence.

5. Verification of Clones

Plasmid DNA for each of the constructs is prepared essentially accordingto Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory,New York (1982). The purified DNA is used in restriction analysis andDNA sequencing across the cloning sites to verify the clones. The insertof pCIB2001/SalChit is in the same orientation as pCIB2001/BamChit andpCIB2001/NcoChit, whose orientation is forced by the cloning methodused.

E. Preparation of a Chimeric Construct

1. Construction of pBSGus1.2

pBSGus1.2 is created by a three part ligation of a 391 bp SalI/SnaBIfragment from pRAJ265 [Jefferson et al., EMBO J. 6: 3091-3907 (1987) andGUS User Manual, Clonetech Labs (Palo Alto, Calif.)] with a 1707 bpSnaBI/EcoRI fragment from pBI221 [Jefferson et al., Id.] and pBSdigested with SalI and EcoRI. Transformants are isolated and analyzed byrestriction digestion and DNA sequencing. One verified clone is namedpBSGus1.2.

2. Construction of pBSChit/GUS

The 2613 bp cucumber chitinase promoter region in pCIB2001/BamChit isisolated using PCR with the GeneAmp kit from Perkin-Elmer/Cetus(Norwalk, Conn.) according to the manufacturer's recommendations andusing the following primers:

5′-GCCTCGAGGATCCTATTGAAAAAG-3′ (SEQ ID No. 82) and

5′-GCCTCGAGTGCTTAAAGAGCTTTC-3′ (SEQ ID No. 83).

The PCR product consists of the 2613 bp promoter with XhoI restrictionenzyme sites on both ends of the sequence due to the design of theprimers. This amplified product is then digested with XhoI and the pieceis gel-purified. The purified fragment is ligated with pBSGus1.2 thathas been digested with XhoI and treated with calf intestinal alkalinephosphatase. Transformants are isolated and analyzed by DNA sequencing.One verified clone, containing the cucumber promoter in its correctorientation in front of the GUS gene, is named pBSChit/GUS.

3. Construction of pCIB2001/Chit/GUS

The plasmid pBSChit/GUS is digested with KpnI and XbaI, and the insertpiece containing the chitinase promoter and GUS gene with its flanking3′ sequences is gel-purified. The purified fragment is ligated withpCIB2001 (Section D, above) that has been digested with KpnI and XbaI.Transformants are isolated on L-kan plates and plasmid DNA is analyzedby restriction digestion and DNA sequencing across the cloning sites.One verified clone is named pCIB2001/Chit/GUS.

Example 63 Isolation of a Genomic Clone Encoding Acidic β-1,3-glucanase

A genomic library is constructed as described in Example 20 and screenedwith a cDNA clone for the acidic β-1,3-glucanase, Example 59. A lambdaclone is isolated, and a 1 kb EcoRI fragment of this lambda clonesubcloned into Bluescript as described above in Example 61. Thebluescript clone is named pBSGL6e.

Example 63A Isolation of a Genomic Clone for Arabidopsis PR-1

The Arabidopsis PR-1 cDNA cloned in pAPR1C-1 (sequence 33) was used as ahybridization probe in screening an Arabidopsis λEMBL 3 genomic library(purchased from Clontech). Four hybridizing plaques were plaque purifiedusing conventional techniques and λ DNA was isolated from each one withlambdasorb (Promega). The λ DNA thus isolated was digested with XhoI,electrophoresed, transferred to hybridization membrane for hybridizationwith the PR-1 cDNA. A fragment of 7 kb was found to hybridize to thecDNA. DNA from one of the four purified plaques was then redigested withXhoI and ligated into the XhoI site of pBluescript (Stratagene). Acolony carrying the promoter fragment was identified by probing witholigonucleotide DC21 from the PR-1 coding sequence (position +110 to+84) and the plasmid contained therein was designated pAtPR1-P anddeposited Jan. 5, 1994 with the the Agricultural Researech CultureCollection, Intenational Depositing Authority, 1815 N. UniversityStreet, Peoria, Ill. 61604 (NRRL deposit no. NNRL B-21169). Restrictionanalysis identified the 7 kb XhoI fragment as extending 4.2 kb upstreamof the ATG of the PR1 gene.

G. CHIMERIC GENES CONTAINING ANTI-PATHOGENIC SEQUENCES

This section describes combinations of cDNA sequences with sequencesthat promote transcription of the cDNA's and with those that facilitateprocessing of the 3′ end of the mRNA in the plant cells.

1. Construction of Plasmids Containing Plant Expression Cassettes.

This first set of examples covers the construction of plasmids whichcontain expression cassettes.

Example 64 Construction of pCGN1509 (tobacco RUBISCO small subunitpromoter cassette)

pCGN1509 is an expression cassette plasniid containing the 5′ regulatoryregion and promoter from a tobacco ribulose-bis-phosphate carboxylase(RuBISCO) small subunit gene, and the 3′ region from the octopinesynthase (ocs) gene of the Ti plasmid of Agrobacterium tumefaciens, withunique restriction sites between these two parts. The 5′ regulatoryregion is ultimately derived from a 3.4 kb EcoRI fragment containingtobacco RuBISCO small subunit gene TSSU3-8 (O'Neal et al., Nucl. AcidsRes. 15:8661-8677 (1987)).

The 3.4 kb EcoRI fragment of TSSU3-8 is cloned into the EcoRI site ofM13 mp18 (Yanisch-Perron et al., Gene 53:103-119 (1985)) to yield an M13clone 8B. Single-stranded DNA is used as a template to extendoligonucleotide primer “Probe 1” (O'Neal et al., Nucl. Acids Res.15:8661-8677 (1987)) using the Klenow fragment of DNA polymerase I.Extension products are treated with mung bean nuclease and then digestedwith HindHIII to yield a 1450 bp fragment containing the small subunitpromoter region; the fragment is cloned into HindHII-SmaI digested pUC18(Yanisch-Perron et al., Gene 53:103-119 (1985)) to yield pCGN625.

The BamHI-EcoRI fragment of pCGN625 is cloned into the large BamHI-EcoRIfragment (plasmid backbone) of BamHI-EcoRI digested pCGN607 in which theSmaI site at position 11207 (Barker et al., Plant Mol Biol 2: 335:350(1983)) of the ocs 3′ region is converted to a BglII site by ligation ofa synthetic BglII linker (Facciotti et al., Bio/Technology 3:241-246(1985)). This yields plasmid pCGN630. The BamHI site of pCGN630 isdeleted by digestion with BamHI and treatment with the Klenow fragmentof DNA polymerase I to create pCGN1502. The KpnI site of pCGN1502 isreplaced by a BamHI site by digestion of pCGN1502 with KpnI, treatmentwith Klenow enzyme, and ligation of a synthetic BamHI linker. Theresulting construction is pCGN1509.

Example 65 Double CAMV 35S Promoter/Terminator Cassette ContainingAmpicillin Resistance and pCGN1431 (A Double CAMV 35SPromoter/Terminator Cassette Containing Chloramphenicol Resistance)

pCGN1761 contains a double CaMV 35S promoter and the tml-3′ region withan EcoRI site between contained in a pUC-derived plasmid backbone. Thepromoter-EcoRI-3′ processing site cassette is bordered by multiplerestriction sites for easy removal. The plasmid is derived by a seriesof steps (see below) from an initial double-35S plasmid, pCGN2113, whichitself is derived from pCGN164, and pCGN638. The plasmid pCGN2113 isdeposited with ATCC March (accession number 40587).

pCGN1431 also contains the double CAMV 35S promoter and the tml 3′region with a multiple cloning site between them. Thispromoter/terminator cassette is contained in a pUC-derived vector whichcontains a chloramphenicol rather than ampicillin resistance gene. Thecassette is bordered by multiple restriction sites for easy removal.

A. Construction of pCGN986

pCGN986 contains a cauliflower mosaic virus 35S (CaMV35) promoter and aT-DNA tml-3′-region with multiple restriction sites between them.pCGN986 is derived from another plasmid, pCGN206, containing a CaMV35Spromoter and a different 3′ region, the CaMV region VI 3′-end. The CaMV35S promoter is cloned as an AluI fragment (bp 7144-7734) (Gardner etal., Nucl. Acids Res. 9: 2871-2888 (1981)) into the HincII site ofM13mp7 (Messing et al., Nucl. Acids Res. 9:309-321 (1981)) to createC614. An EcoRI digest of C614 produced the EcoRI fragment from C614containing the 35S promoter which is cloned into the EcoRI site of pUC8(Vieira and Messing, Gene 19:259-268 (1982)) to produce pCGN147.

pCGN148a containing a promoter region, selectable maker (Kanamycin with2 ATG's) and 3′ region, is prepared by digesting pCGN528 with BglII andinserting the BamHI-BglHII promoter fragment from pCGN147. This fragmentis cloned into the BglII site of pCGN528 so that the BglII site isproximal to the kanamycin gene of pCGN528.

The shuttle vector, pCGN528, used for this construct is made as follows:pCGN525 is made by digesting a plasmid containing Tn5 which harbors akanamycin gene (Jorgensen et al., Mol. Gen. Genet. 177:65 (1979)), withHindIII-BamHI and inserting the HindIII-BamHI fragment containing thekanamycin resistance gene into the HindIII-BamHI sites in thetetracycline gene of pACYC184 (Chang and Cohen, J. Bacteriol.134:1141-1156 (1978)) pCGN526 is made by inserting the BamHI fragment 19of pTiA6 (Thomashow et al., Cell 19:729-739 (1980)) modified with XhoIlinkers inserted into the SmaI site, into the BamHI site of pCGN525.pCGN528 is obtained by deleting the small XhoI and religating.

pCGN149a is made by cloning the BamHI Kanamycin gene fragment frompMB9KanXXI into the BamHI site of pCGN148a. pMB9KanXXI is a pUC4Kvariant (Vieira and Messing, Gene 19:259-268 (1982)) which has the Xholsite missing but contains a function kanamycin gene from Tn903 to allowfor efficient selection in Agrobacterium.

pCGN149a is digested with HindII and BamHI and ligated to pUC8 digestedwith HindIII and BamHI to produce pCGN169. This removes the Tn903kanamycin marker. pCGN565 and pCGN169 are both digested with HindIII andPstl and ligated to form pCGN203, a plasmid containing the CaMV 35Spromoter and part of the 5′-end of the TN5 kanamycin gene (up to thePstl site, (Jorgensen et al. Mol. Gen. Genet. 177: 65 (1979)). A 3′regulatory region is added to pCGN203 from pCGN204 (an EcoRI fragment ofCaMV (bp 408-6105) containing the 3′ region of gene VI subcloned intopUC18 (Gardner et al., Nucl. Acids Res. 9: 2871-2888 (1981)) bydigestion with HindIII and Pstl and ligation. The resulting cassette,pCGN206, is the basis for the construction of pCGN986.

The pTiA6 T-DNA tml 3′-sequences are subcloned from the Bam19 T-DNAfragment (Thomashow et al., Cell 19:729-739 (1980)) as a BamHI-EcoRIFragment (nucleotides 9062 to 12, 823, numbering as in (Barker et al.,Plant Mo. Biol. 2: 335-350 (1983)) and combined with the pACYC184 (Changand Cohen, J. Bacteriol. 134:1141-1156 (1978)) origin of replication asan EcoRI-HindII fragment and a gentamycin resistance marker (fromplasmid pLB41), (D. Figurski) as a BamHI-HindII fragment to producepCGN417.

The unique SmaI site of pCGN417 (nucleotide 11,207 of the Bam19fragment) is changed to a SacI site using linkers and the BamHI-SacIfragment is subcloned into pCGN565 to give pCGN971. The BamHI site ofpCGN971 is changed to an EcoRI site using linkers to yield pCGN971E. Theresulting ECoRI-SacI fragment of pCGN971E, containing the tml 3′regulatory sequences, was joined to pCGN206 by digestion with EcoRI andSacI to give pCGN975. The small part of the Tn5 kanamycin resistancegene is deleted from the 3′-end of the CaMV 35S promoter by digestionwith SalI and BglII, blunting the ends and ligation with SalI linkers.The final expression cassette pCGN986 contains the CaMV 35S promoterfollowed by two SalI sites, an XbaI site, BamHI, SmaI Kpnl and the tml3′region (nucleotides 11207-9023 of the T-DNA).

B. Construction of pCGN164

The AluI fragment of CaMV (1)p 7144-7735) (Gardner et al., Nucl. AcidsRes. 9: 2871-2888 (1981)) is obtained by digestion with AluI and clonedinto the HincII site of M13mp7 (Vieira and Messing, Gene 19: 259-268(1982)) to create C614. An EcoRI digest of C614 produces the EcoRIfragment from C614 containing the 35S promoter which is cloned into theEcoRI site of pUC8 (Vieira and Messing, Gene 19: 259-268 (1982)) toproduce pCGN146. To trim the promoter region, the BglII site (bp7670) istreated with BglII and Bal31 and subsequently a BglII linker is attachedto the Bal31 treated DNA to produce pCGN147. pCGN147 is digested withEcoRI HphI and the resultant EcoRI-HphI fragment containing the 35Spromoter is ligated into EcoRI-SmalI digested M13mp8 (Vieira andMessing, Gene 19:259-268 (1982)) to create pCGN164.

C. Construction of pCGN638

Digestion of CaMV10 (Gardner et al., Nucl. Acids Res. 9: 2871-2888(1981)) with BglII produces a BglII fragment containing a 35S promoterregion (bp 6493-7670) which is ligated into the BamHI site of pUC19(Norrander et al., Gene 26:101-106 (1983)) to create pCGN638.

D. Construction of pCGN2113

pCGN164 is digested with EcoRV and BamHI to release a EcoRV-BamHIfragment which contained a portion of the 35S promoter (bp 7340-7433);pCGN638 is digested with HindIII and EcoRV to release a HindIII-EcoRVfragment containing a different portion of the 35S promoter (bp6493-7340). These two fragments are ligated into pCGN986 which has beendigested with HindII and BamHI to remove the HindIII-BamHI fragmentcontaining the 35S-promoter, this ligation produces pCGN639, whichcontains the backbone and tml-3′ region from pCGN986 and the two 35Spromoter fragments from pCGN164 and pCGN638. pCGN638 is digested withEcoRV and DdeI to release a fragment of the 35S promoter (bp 7070-7340);the fragment is treated with the Klenow fragment of DNA polymerase I tocreate blunt ends, and is ligated into the EcoRV site of pCGN639 toproduce pCGN2113 having the fragment in the proper orientation.

E. Construction of pCGN1761

pCGN2113 is digested with EcoRI and the plasmid is ligated in thepresence of a synthetic DNA adaptor containing an XbaI site and a BamHIsite (the adaptor contains EcoRI sticky ends on either end, but theadjacent bases are such that an EcoRI site is not reconstructed at thislocation) to produce pCGN2113M. pCGN2113M is digested to completion withSacI and then subjected to partial digestion with BamHI. This DNA isthen treated with T4 DNA polymerase to create blunt ends and an EcoRIlinker is ligated into the blunt-ended plasmid. After transformation aplasmid clone which contains a unique EcoRI site between the promoterand the intact tml-3′ region is selected and designated pCGN1761.

F. Construction of pCGN1431

The SalI-EcoRI fragment of pCGN2113, which contains the entirepromoter-polylinker-3′ cassette, is removed by Sall-EcoRI digestion andcloned into SalI-EcoEl digested pCGN565 to create pCGN2120; pCGN565 is acloning vector based on pUC8-Cm (K. Buckley, PH.D. Thesis, UC San Diego1985), but containing the polylinker from pUC18 (Yanisch-Perron et al.,Gene 53: 103-119 (1985)). pCGN2120 is digested to completion with PstIand then religated. A clone is selected which had deleted only the 858bp PstI-PstI fragment (9207-10065, Barker et al., 1983, supra) from thetml 3′ region to create pCGN1431.

2. Chimeric Genes

This second section describes the subcloning of cDNA's into thecassettes in both sense and anti-sense orientation.

Example 66 Construction of pCGN1752A and pCGN1752B (SSU Promoter/PR-1AExpression Cassette; Sense and Anti-Sense Orientation))

The 807 bp EcoRI fragment of pBSPR1-207 is subcloned into EcoRI digestedpCGN1509 and plasmids bearing the cDNA in each of the two possibleorientations are selected; a plasmid in which the tobacco RuBISCO smallsubunit promoter would be expected to generate a transcript with themRNA sense strand of PR1a is designated pCGN1752A, and a plasmid inwhich the tobacco RuBISCO small subunit promoter would be expected togenerate a transcript with the anti-sense strand (i.e. complementarysequence of the mRNA) of PR1a is designated pCGN1752B.

Example 67 Construction of pCGN1753A and pCGN1753B (SSU Promoter/PR-1BExpression Cassette; Sense and Anti-Sense Orientation)

The 717 bp EcoRI fragment of pBSPR1-1023 is subcloned into EcoRIdigested pCGN1509 and plasmids bearing the cDNA in each of the twopossible orientation are selected; a plasmid in which the tobaccoRuBISCO small subunit promoter would be expected to generate atranscript with the mRNA sense strand of PR-1b is designated pCGN1753A,and a plasmid in which the tobacco RuBISCO small subunit promoter wouldbe expected to generate a transcript with the antisense strand (i.e.complementary sequence of the mRNA) of PR-1b is designated pCGN1753B.

Example 68 CAMV 35S Promoter/PR-1A Expression Cassette (Sense andAnti-Sense Orientation))

A 807 bp EcoRI fragment bearing a tobacco PR1a cDNA is released frompBSPR1-207 by EcoRI digestion and subcloned into EcoRI digested pCGN565to yield pCGN1750. A 717 bp EcoRI fragment bearing the entire codingregion of a tobacco PR-1b cDNA is released from pBSPR1-1023 by digestionwith EcoRI and subcloned into EcoRI digested pCGN565 to yield pCGN1751.These two plasmids are constructed to facilitate subsequent subcloningexperiments.

The 807 bp EcoRI fragment of pCGN1750 is subcloned into EcoRI digestedpCGN1761 and plasmids bearing the cDNA in each of the two possibleorientation are selected; a plasmid in which the double 35S promoterwould be expected to generate a transcript with the mRNA sense strand ofPR-1a is designated pCGN1762A, and a plasmid in which the double 35Spromoter would be expected to generate a transcript with the anti-sensestrand (i.e. complementary sequence of the mRNA) of PR-1a is designatedpCGN1762B.

Example 69 Construction of pCGN1763A and pCGN1763B (Double CaMV 35SPromoter/PR-1b Expression Cassette; Sense and Anti-Sense Orientation)

The 717 bp EcoRI fragment of pCGN1751 (see above) is subcloned intoEcoRI digested pCGN1761 and plasmids bearing the cDNA in each of the twopossible orientation are selected; a plasmid in which the double 35Spromoter would be expected to generate a transcript with the mRNA sensestrand of PR-1b is designated pCGN1763A, and a plasmid in which thedouble 35S promoter would be expected to generate a transcript with theanti-sense strand (i.e. complementary sequence of the mRNA) of PR-1b isdesignated pCGN1763B.

Example 70 Construction of pCIB1002 and pCIB1003 (Double CAMV 35SPromoter/PR-R major Expression Cassettes; Sense and Anti-SenseOrientation)

The plasmid pBSPRR-401 is partially digested with EcoRI and theresulting DNA fragments are separated on a 1.0% low-gelling temperatureagarose gel. A band at about 900 base pairs, which contains the fulllength PR-R cDNA insert, is excised and ligated to pCGN1761 which hadbeen digested to completion with EcoRI and dephosphorylated using calfintestine alkaline phosphatase. The DNA is ligated and transformed asdescribed above, positive colonies are screened and their plasmids areanalyzed. One plasmid which contains the PR-R cDNA in a senseorientation relative to the double CAMV 35S promoter, is selected anddesignated as pCIB1002. A second plasmid, in which the PR-R cDNA is inan anti-sense orientation relative to the double CAMV 35S promoter isselected and designated pCIB1003.

Example 71 Construction of pCIB1020 and pCIB1021 (Double CAMV 35SPromoter/PR-P Expression Cassette; Sense and Anti-Sense Orientation)

The plasmid pBScht28 (see above) is digested with EcoRI and fragmentsare separated on a 0.5% LGT agarose gel. The band containing the PR-PcDNA is excised and mixed with pCGN1761 (see above) which had beendigested with EcoRI, treated with calf intestinal alkaline phosphatase(CIAP) and purified on a 0.5% low gelling temperature (LGT) agarose gel.The mixture is ligated and transformed as described. Plasmids arescreened for insertion of the PR-P cDNA in either orientation. Oneplasmid, in which the PR-P cDNA is inserted in a sense orientationrelative to the double CAMV 35S promoter, is designated pCIB1020. Aplasmid in which the PR-P cDNA is inserted in an anti-sense orientationrelative to the double CAMV 35S promoter is designated pCIB1021.

The plasmid pBScht28 is deposited with ATCC (accession number 40588).

Example 72 Construction of pCIB1022 and pCIB1023 (Double CAMV 35SPromoter/PR-Q Expression Cassette; Sense and Anti-Sense Orientation)

The full-length cDNA sequence for PR-Q is contained on two differentplasmids, PBScht15 contains the 3′ end of the cDNA and pBScht′-4contains the 5′ end of the cDNA. Because of this situation, the plasmidspCIB1022 and pCIB1023 are constructed in a three way ligation and differby their orientation in the pCGN1761 vector. pCGN1761 is digested withEcoRI, treated with CIAP and purified on a 0.5% LGT agarose gel.pBScht15 (see above) is digested with NsiI and EcoRI and the fragmentsare separated on a 0.5% LGT agarose gel. A PCR reaction using pBScht5′-4(see above) as a template and oligonucleotides:

1) 5′CTATGAATGCATCATAAGTG 3′ (SEQ ID No. 84); and

2) 5′GCGGAATTCAAAAAAAAAAAAAAACATAAG 3′ (SEQ ID No. 85)

as primers is performed to amplify the 5′ end of the PR-Q cDNA. The PCRproduct is purified and digested with NsiI and EcoRI and the 210 bpproduct is purified from a 1.0% LGT agarose gel. The purified pCGN1761vector, the 810 bp NsiI/EcoRI fragment from pBScht15 and the NsiI/EcoRIdigested PCR fragment are ligated and transformed as described above.Transformants are screened and selected which include the entire cDNA ineither orientation.

One plasmid which has the full-length PR-Q cDNA in a sense orientationrelative to the double CAMV 35S promoter is designated as pCIB1022. Aplasmid in which the full-length PR-Q cDNA is inserted in the anti-senseorientation relative to the promoter is designated pCIB1023.

Example 73 Construction of pCIB1024 and pCIB1025 (Double CAMV 35SPromoter/PR-O′ Expression Cassette; Sense and Anti-Sense Orientation)

The plasmid pBSGL6e is deposited with ATCC (accession number 40535). ThePR-O′ cDNA cloned into pBSGL6e is truncated at the 5′ end and is missingalmost all of the complete signal sequence. This sequence is necessaryfor extracellular transport of the protein and should be replaced inorder to engineer transgenic plants that secrete the protein. Thisexample describes the subcloning of the PR-O′ cDNA into the double CAMV35S expression cassette in such a way that a signal peptide from PR-1ais added to the PR-O′ protein.

This construction is carried out as a complicated three-way ligation.First a fusion of the PR-1a leader and signal peptide and the codingsequence of the mature PR-O′ is made by a PCR gene fusion method. Thenthis piece is ligated along with the 3′ end of the PR-O′ cDNA into thepCGN1761 vector and transformants are selected with the insert in eitherorientation relative to the promoter.

A gene fusion technique based on PCR amplification has been developed byHo, S. et al, Gene 77: 51-59 (1989). In this technique a gene fusion ismade by creating two fragments with overlapping ends by PCR. In asubsequent reaction these two fragments are then fused also by PCR togenerate a perfect fusion between the two molecules. This strategy isused to fuse the PR-1a signal peptide and leader to the PR-O′ cDNA. Fouroligonucleotides are synthesized with the following sequences:

GP50—5′CCATAACAAACTCCTGCTTGGGCACGGCAAGAGTGGGATA 3′ (SEQ ID No. 86)

GP51—5′TATCCCACTCTTGCCGTGCCCAAGCAGGAGTTTGTTATGG 3′ (SEQ ID No. 87)

GP52—5′GATCGAATTCATTCAAGATACAACATTTCT 3′ (SEQ ID No. 88)

GP53—5′CATTCRGAAGGTCCGG 3′ (SEQ ID No. 89).

The GP50 and GP51 oligonucleotides are complementary to each other andcontain the DNA sequence desired for the fusion between the PR-1a leaderand signal and the PR-O′ mature coding sequence. This is diagrammedbelow:

GP51 5′ TATCCCACTCTTGCCGTGCCCAAGCAGGAGTTTGTTATGG 3′   3′ ATAGGGTGAGAACGGCACGGGTTCGTCCTCAAACAATACC 5′ GP5O      |  PR-1A    ||  PR-O′  |

The oligonucleotide GP52 is the same sequence as the 5′ end of the PR-1acDNA and it contains on the 5′ end a sequence encoding an EcoRI site:

(SEQ ID No. 88) GP52 - 5′ GATCGAATTCATTCAAGATACAACATTTCT 3′       |EcoRI| PR-1a  |

The oligonucleotide GP53 serves as a primer and is complementary topositions 180 to 195 of the PR-O′ sequence m SEQ ID No. 13.

In order to fuse the two pieces of DNA two PCR reactions are set up. Oneuses the plasmid pBSPR1-207 as a template and the two primers GP52 andGP50; the other uses pBSGL6e as a template and the primers GP51 andGP53. The PCR products are analyzed by gel electrophoresis and it isdetermined that the reactions are successful.

The PCR products are then purified and an aliquot of each is used in asecond stage PCR reaction. In this reaction the templates are both ofthe products from the first two reactions and the primers are GP52 andGP53. A modified PCR reaction is established such that in the firstround of synthesis the DNA templates are added without the primers andthe templates are heated and allowed to cool and then extended at 65° C.The two primers are then added and the PCR reaction is carried outnormally.

An aliquot of the PCR reaction is analyzed by gel electrophoresis and itis determined that the reaction is successful. The remaining DNA is thenpurified and digested with SacI and EcoRI and the digest iselectrophoresed on a 1.5% LGT agarose gel. The band corresponding to thecorrect PCR product is excised and used for ligation.

The plasmid pBSGL6e is digested with both SacI and EcoRI and the digestis electrophoresed on a 0.5% LGT agarose gel. The 1.0 kb band containingthe large PR-O′ fragment is excised and ligated with the SacI-EcoRI PCRproduct from above and the plasmid pCGN1761 which had been digested withEcoRI and CIAP and purified on a 0.5% LGT agarose gel. The fragments areligated and transformed as described above. Transformants are screenedfor the correct construct.

A plasmid in which the PR-1a leader and signal has been fused to thePR-O′ mature coding sequence and is in the sense orientation relative tothe promoter is designated pCIB 1024; this construct is confirmed by DNAsequencing.

A plasmid with the correct fusion but in the opposite orientationrelative to the promoter is designated pCIB1025. This construct is alsoverified by DNA sequencing.

Example 74 Construction of pCIB1024A and pCIB1025A (Double 35SPromoter/PR-O′ Expression Cassette; Sense and Ant-Sense Orientation)

The plasmid pBSGL5B-12, which contains a full-length cDNA encoding thePR-O′ protein is digested with EcoRI and the fragments are separated ona 0.5% LGT agarose gel. The 1.2 kb band is excised and ligated withpCGN1761 which is digested with EcoRI, treated with CIAP and purified ona 0.5% LGT agarose gel as described above. The ligation mixture istransformed as described and transformants are screened for a clonecontaining the PR-O′ cDNA in either orientation. One plasmid, in whichthe cDNA is inserted in a sense orientation relative to the double CAMV35S promoter, is designated pCIB 1024. A plasmid in which the cDNA isinserted in an anti-sense orientation relative to the promoter isdesignated as pCIB1025A.

Example 75 Construction of pCIB1032 and pCIB1033 (Double 35SPromoter/PR-2 Expression Cassette; Sense and Anti-Sense Orientation)

The plasmid pBSGL117 contains a full-length cDNA encoding the PR-2protein. The PR-2 cDNA from pBSGL117 is subcloned into the pCGN1761expression plasmid in either orientation to create pCIB1032 andpCIB1033. However, the cDNA contains an internal EcoRI site and so thecDNA has to be excised by a partial EcoRI digest.

The plasmid pBSGL117 is digested with EcoRI under conditions in which apartial digest resulted. The digestion products are separated on a 0.5%LGT agarose gel and the 1.2 kb band containing the full-length cDNA forPR-2 is excised and ligated to pCGN1761 which had been digested withEcoRI, treated with CIAP and purified on a 0.5% LGT agarose gel. Theligation and transformation are carried out as previously described.Positive transformants are isolated and screened for the presence of thelarge PR-2 cDNA fragment inserted in either orientation. One plasmid,with the PR-2 cDNA subcloned in a sense orientation relative to thetranscriptional start site is designated as pCIB1032 and a plasmid withthe fragment in an anti-sense orientation is designated as pCIB1033. Thestructure of these constructs is verified by DNA sequencing.

Example 76 Construction of pCIB1034 and pCIB1035 (Double 35SPromoter/PR-N Expression Cassette; Sense and Anti-Sense Orientation)

A plasmid containing a full-length cDNA encoding the PR-N protein isused as a source to subclone the PR-N cDNA into the pCGN1761 expressionplasmid in either orientation. The resulting plasmids are designated aspCIB1034 and pCIB1035. However, the cDNA contains an internal EcoRI siteand so the cDNA has to be excised by a partial EcoRI digest.

The plasmid containing the full-length PR-N cDNA is digested with EcoRIunder conditions in which a partial digest results. The digestionproducts are separated on a 0.5% LGT agarose gel and the 1.2 kb bandcontaining the full-length cDNA for PR-N is excised and ligated topCGN1761 which has been digested with EcoRI, treated with CIAP andpurified on a 0.5% LGT agarose gel. The ligation and transformation iscarried out as previously described.

Positive transformants are isolated and screened for the presence of thelarge PR-N cDNA fragment inserted in either orientation. One plasmid,with the PR-N cDNA subcloned in a sense orientation relative to thetranscriptional start site is designated as pCIB1034 and a plasmid withthe fragment in an anti-sense orientation is designated as pCIB1035. Thestructure of these constructs is verified by DNA sequencing.

Example 77 Construction of pCIB1036 and pCIB1037 (Double 35SPromoter/PR-O Expression Cassette; Sense and Anti-Sense Orientation)

A plasmid containing a full-length cDNA encoding the PR-O protein isused as a source to subclone the PR-O cDNA into the pCGN1761 expressionplasmid in either orientation. The resulting plasmids are designated aspCIB1036 and pCIB1037. However, the cDNA contains an internal EcoRI siteand so the cDNA has to be excised by a partial EcoRI digest.

The plasmid containing the full-length PR-O cDNA is digested with EcoRIunder conditions in which a partial digest results. The digestionproducts are separated on a 0.5% LGT agarose gel and the 1.2 kb bandcontaining the full-length cDNA for PR-O is excised and ligated topCGN1761 which has been digested with EcoRI, treated with CIAP andpurified on a 0.5% LG3T agarose gel. The ligation and transformation iscarried out as previously described. Positive transformants are isolatedand screened for the presence of the large PR-O cDNA fragment insertedin either orientation. One plasmid, with the PR-O cDNA subcloned in asense orientation relative to the transcriptional start site isdesignated as pCIB1036 and a plasmid with the fragment in an anti-senseorientation is designated as pCIB1037. The structure of these constructsis verified by DNA sequencing.

Example 78 Construction of pCIB1038 and pCIB1039 (Double 35Spromoter/PR-2′ Expression Cassette; Sense and Anti-Sense Orientation)

A plasmid (pBSGL135 from Example 48) containing a full-length cDNAencoding the PR-2′ protein is used as a source to subclone the PR-2′cDNA into the pCGN1761 expression plasmid in either orientation. Theresulting plasmids are designated as pCIB1038 and pCIB1039. However, thecDNA contains an internal EcoRI site and so the cDNA has to be excisedby a partial EcoRI digest.

The plasmid containing the full-length PR-2′ cDNA is digested with EcoRIunder conditions in which a partial digest results. The digestionproducts are separated on a 0.5% LGT agarose gel and the 1.2 kb bandcontaining the full-length cDNA for PR-2′ is excised and ligated topCGN1761 which has been digested with EcoRI, treated with CIAP andpurified on a 0.5% LGT agarose gel. The ligation and transformation iscarried out as previously described. Positive transformants are isolatedand screened for the presence of the large PR-2′ cDNA fragment insertedin either orientation. One plasmid, with the PR-2′ cDNA subcloned in asense orientation relative to the transcriptional start site isdesignated as pCIB1036 and a plasmid with the fragment in an anti-senseorientation is designated as pCIB1039. The structure of these constructsis verified by DNA sequencing.

The foregoing methods can be used to construct a double 35Spromoter-driven expression cassette for any other cDNA sequenceisolated, including, but not limited to, those cDNAs presented in SEQ IDNo. 26 (PR-2″), SEQ ID No. 29 (basic tobacco chitinase/lysozyme), SEQ IDNo. 30 (acidic tobacco chitinase/lysozyme), and SEQ ID Nos. 31 and 32(PR-4a and PR-4b).

Example 79 Construction of pCIB1005B and pCIB1006B (Double CAMV 35SPromoter/Basic Glucanase Expression Cassette; Sense and Anti-SenseOrientation)

The plasmid pGLN17 is a hybrid cDNA encoding the basic β 1,3 glucanasefrom Nicotiana tabacum (Shinshi, H. et al., 1988 supra) constructed byfusing the 5′ end of the pGL31 clone and the 3′ end of the pGL36 clone.The sequence encoded in this hybrid cDNA is shown in SEQ ID No. 20. Itis found that this cDNA is truncated at the 5′ end and does not encodethe entire signal peptide. In order to make transgenic plants in whichthis protein is properly targeted (ie, the central vacuole), it isnecessary to add this sequence back on to the truncated cDNA. Therefore,the double CAMV 35S expression cassette is constructed in a two stepprocess. In the first step the signal peptide of the cDNA is replaced bya signal peptide encoded in the genomic clone. In the second step, this“repaired cDNA” is moved into the expression vector.

The plasmid pSGL2 is a subclone of the pGLN17cDNA. This plasmid isdigested with ClaI and EcoRI and the 1 kb fragment containing theglucanase cDNA is isolated from a LGT agarose gel. The pBluescriptplasmid is digested with EcoRI, treated with CIAP and purified on a LGTagarose gel.

The plasmid pBS-Gluc 39.1 (ATCC accession no. 40526) contains a 4.4 kbinsert which includes the glucanase coding sequence, about 1.5 kb of 5′flanking sequence, a 600 bp intron, and about 1 kb of 3′ flankingsequence. This plasmid is used as a template in a PCR experimentcontaining the following two primers:

A. 5′CATCTGAATTCTCCCAACAAGTCTTCCC 3′ (SEQ ID No.90)

B. 5′AACACCTATCGATTGAGCCCCTGCTATGTCAATGCTGGTGGC 3′ (SEQ ID No. 91)

The result of this amplification is to produce a fragment to replace thetruncated 5′ end of the glucanase cDNA. A single-base mutation creatingan EcoRI site is introduced to facilitate cloning experiments. The PCRproduct is digested with EcoRI and ClaI and fragments are separated on a2.0% LGT agarose gel. A 120 bp band is excised, mixed with the 1 kbClaI-EcoRI fragment from pSGL2 and the purified, EcoRI digestedbluescript vector, ligated and transformed as described above.Transformants are screened for the presence of the insert and oneplasmid with the proper structure is designated pCIB1009.

The plasmid pCIB1009 is digested with EcoRI and the 1.2 kb fragment ispurified on a LGT agarose gel. The plasmid pCGN1761 is digested withEcoRI, treated with CIAP, purified on a LGT agarose gel, mixed with the1.2 kb EcoRI fragment, and then ligated and transformed. Transformantsare screened for the presence of the insert. One plasmid, in which theglucanase cDNA is in a sense orientation relative to the CAMV promoteris designated as pCIB1005B. Another plasmid, with the cDNA insert in ananti-sense orientation is designated pCIB1006B.

Example 80 35S Promoter/Bask Chitinase Expression Cassette (Sense andAnti-Sense Orientation)

The plasmid pSCH10 contains a cDNA insert of the tobacco basic chitinasewhich is similar to the insert in pCHN50 (Shinshi, H. et al., supra) butwith an extension of 81 base pairs on the 5′ end. The 80 extra basepairs are:

5′GGATCCGTTTGCATTTCACCAGTTTACTACTACATTAAAATGAGGCTTTGTAAATTC  |   |    |    |    |   |   1   10     20    30   40    505′ACAGCTCTCTCTTCTCTACTATTT 3′ (SEQ ID No. 92)     |    |      60    70    80

pSCH10 is digested with BamHI and then ligated with a molecular adaptorwith the sequence 5′GATCCGGAATTCCG 3′ as described in Example 5 above.The ligation product is then purified and digested with EcoRI and the1.2 kb fragment containing the adapted chitinase cDNA is purified from aLGT agarose gel. This fragment is mixed with EcoRI digested, CIAPtreated pCGN1761 which is also purified from a LGT agarose gel and themixture is ligated and transformed. Transformants are screened for thechitinase cDNA insert and one plasmid which contains the chitinase cDNAin a sense orientation relative to the CAMV promoter is designated aspCIB1007. A plasmid with the chitinase cDNA in an anti-sense orientationwith respect to the promoter is designated as pCIB1008.

Example 81 Construction of pCGN1788A and pCGN1788B (Double CAMV 35SPromoter/SAR8.2 Expression Cassette; Sense and Anti-Sense Orientation)

The plasmids pCIB/SAR8.2a and pCIB/SAR8.2b are deposited with ATCC,accession numbers 40584 and 40585 respectively. The pSAR8.2a cDNA (seeabove) is subcloned into the double CAMV 35S promoter/3′tml terminatorcassette pCGN1431 (see above) using a PCR amplification method. Fouroligonucleotides, two for each of the sense and anti-sense constructionsand each one 33 nucleotides in length, are synthesized for use asprimers to generate the cDNA sequence of pSAR8.2a by PCR using theplasmid pSAR8.2a as template. The primers contained additional sequencesat their 5′ ends that generated new restriction sites upon completion ofPCR amplification.

For the sense construction the sequence of oligonucleotide 1167 is

5′-GTGACCGAGCTCAAAGAAAAATACAGTACAATA-3′ (SEQ ID No.93)

which generates an SstI site proximal to the 3′ end of the cDNAsequence. The sequence of oligonucleotide 1168 is

5′ACCGTGGGATCCACAGTAAAAAACTGAAACTCC-3′ (SEQ ID No. 94)

and generates a BamHI site proximal to the 5′ end of the cDNA.

For the anti-sense construction, the sequence of oligonucleotide 1224 is

5′-GTGACCGGATCCAAAGAAAAATACAGTACAATA-3′ (SEQ ID No.95)

which generates a BamHI site proximal to the 3′ end of the cDNA. Thesequence of oligonucleotide 1225 is

5′-ACCGTGGAGCTCACAGTAAAAAACTGAAAGTCC-3′ (SEQ ID No. 96)

and generates an SstI site proximal to the 5′ end of the cDNA.

Oligonucleotide 1167 and 1168 are used in a PCR reaction in which theplasmid pSAR8.2a served as a DNA template. The purified PCR productgenerated in this reaction is digested with SstI and BamHI and clonedinto pCGN1431 which is digested with SstI and BamHI. The DNA istransformed and plasmids are screened for the presence of the pSAR8.2acDNA insert in a sense orientation relative to the double CAMV 35Spromoter. Putative plasmids are then subjected to DNA sequencing andone, which has the proper orientation and contains no introducedmutations, is designated pCGN1788A.

For the anti-sense construct, oligonucleotides 1224 and 1225 are used ina PCR reaction as described above. After digestion with SstI and BamHIthe DNA is cloned into SstI and BamHI digested pCGN1431. Putativepositive plasmids are screened for the insertion of the cDNA in ananti-sense orientation relative to the promoter and the constructs areverified by sequencing the entire cDNA. One plasmid, in which the cDNAis inserted in the correct orientation and the DNA sequence is correct,is designated pCGN1788B.

Example 82 Construction of pCIB1000 and pCIB1001 (Double CAMV 35SPromoter/Cucumber Chitinase/Lysozyme Expression Cassette (Sense andAnti-Sense Orientation)

The plasmid pBScucchi/chitinase (ATTC accession number 40528) isdigested with EcoRI and the fragments are separated on a 0.5% LGTagarose gel. The 1.2 kb band is excised and ligated with pCGN1761 whichhad been digested with EcoRI, treated with CIAP and purified on a 0.5%LGT agarose gel as described above. The ligation mixture is transformedas described and transformants are screened for containing thechitinase/lysozyme cDNA in either orientation. One plasmid, in which thecDNA is inserted in a sense orientation relative to the double CAMV 35Spromoter, is designated pCIB1000. A plasmid in which the cDNA isinserted in an anti-sense orientation relative to the double CAMV 35Spromoter is designated pCIB1001.

Example 82A Fusion of the Arabidopsis PR-1 Promoter to the FireflyLuciferase Gene

Plasmid pDO432 containing a gene encoding luciferase (LUC) from fireflywas received from Dr David Ow (University of Califomia, San Diego; se Owet al., 1986; Science 234: 856). The LUC gene was excised from pDO432 bydigestion with XbaI (at position +45 relative to the ATG) and SstI(approximately 1.8 kb downstream of the ATG and outside the LUC codingregion). Additionally, an EcoRI-XbaI promoter fragment was excised frompAtPR1-P; this fragment was 1.4 kb in size and delineated by an XbaIsite 2.8 kb upstream of the PR-1 ATG and an EcoRI site in thepBluescript polylinker distal to the 5′ end of the cloned promoterfragment (at −4.2 kb relative to the ATG). These two fragments werecloned by threeway ligation into EcoRI/SacI cleaved pBluescript thusorienting the LUC gene adjacent to the upstream PR-1 promoter fragment(pAtPR1-Q).

Subsequently, pAtPR1-P was cleaved with XbaI (at the −2.8 kb positionand within the pBluescript polylinker) and religated to generate a PR-1genomic construct without the upstream 1.4 kb promoter fragment andwhich thus ended 2.8 kb upstream of the PR-1 ATG (pAtPR1-R). Thisplasmid was used as a template in PCR with a left-to-right “topstrand”primner extending from positions −237 to −214 (DC39) upstream of thePR-1 ATG (oligo A) and a right-to-left “bottomstrand” primer comprising15 bp of LUC coding sequence extending up to the LUC ATG and a further19 bp of PR-1 sequence extending from the ATG into the PR-1 untranslatedleader (oligo B: sequence: TTT GGC GTC TTC CAT TTT TCT AAG TTG ATA ATGG). This PCR reaction was undertaken for five cycles at 94_C (30 s),40_C (60 s), and 72_C (30 s) followed by 25 cycles at 94_C (30 s), 55_C(60 s) and 72_C (30 s) and this generated a product of 245 bp throughannealing of the homologous PR-1 sequences; the fragment included aBglII site at its left end from the PR-1 promoter. A second PCR reactionwas done using plasmid pDO432 as a template and using a left-to-right“topstrand” oligonucleotide which comprised 15 bp of PR-1 untranslatedleader up to the PR-1 ATG and a further 12 bp of LUC sequence from theATG into the LUC coding sequence (oligo C: sequence: TAT CAA CTT AGA AAAATG GAA GAC GCC AAA) and a right-to-left “bottom strand” oligonucleotideextending from positions 332 to 312 (DC53) into the LUC coding sequence(oligo D). This PCR reaction was done under the same conditions as theone described above and generated a fragment of approximately 300 bpthrough annealing of the homologous LUC sequences; this fragmentincluded a PstI site at its right end, derived from the LUC sequenceamplified.

The two PCR fragments generated above were gel purified using standardprocedures to remove oligonucleotides and were then themselves mixed ina further PCR reaction (“inside-outside PCR”) with oligonucleotides Aand D as primers. Conditions for this reaction were the same asdescribed above. The amplified fragment was a fusion of the PR-1promoter fragment from the first PCR reaction described above and theLUC 5′ Coding sequence from the second PCR reaction described above andhad a BglII site at its left end and a PstI site at its right end. Thefragment was gel purified and cleaved with BglII and PstI to yield aproduct of 545 bp in size which was cloned into pAtPR1-R which hadpreviously been cleaved with the same enzymes. Cleavage of the resultantplasmid (pAtPR1-S) with XbaI released a PR-1 promoter fragment extendingfrom −2.8 kb to the XbaI site downstream of the LUC ATG, the fusionpoint between the PR-1 promoter and the LUC coding sequence being at theATG. This fragment was cloned into XbaI cleaved pAtPR1-Q regeneratingthe full-length PR-1 promoter (4.2 kb) in operational fusion to LUC(pAtPR1-R).

A. Transfer of the Arabidopsis PR-1 Promoter—Firefly Luciferase GeneFusion to pCIB200

pAtPR1-R was cleaved with XhoI and SacI and transferred to SalI/SacIcleaved pCIB200 to create a binary vector construction (pAtPR1-S)suitable for Arabidopsis transformation (see example 28). pAtPR1-S wasthen transferred to Agrobacterium tumefaciens strain A136/pCIB542 fortransfer to Arabidopsis Dijon-O using the method described by Wen-junand Forde, Nucl. Acids Res. 17: 8385-8386 (1989). T2 or T3 linescarrying the PR1-LUC transgene in the homozygous state were generatedfor chemical induction analysis.

H. VECTORS CONTAINING ANTI-PATHOGENIC SEQUENCES

This section details the construction of binary vectors containing allof the chimeric genes.

1. Binary Vectors

This section explains the development of the binary vectors to be used.

Example 83 Construction of pCGN783

pCGN783 is a binary plasmid containing the left and right T-DNA bordersof Agrobacterium tumefaciens octopine Ti-plasmid pTiA6 (Currier andNester, J. Bact 126:157-165 (1976)) the gentamycin resistance gene ofpPh1JI (Hirsch and Beringer, Plasmid 12: 139-141 (1984)), the 35Spromoter of cauliflower mosaic virus (CaMV) (Gardner et al., Nucl. AcidsRes. 9: 2871-2888 (1981)), the kanamycin resistance gene of Tn5(Jorgensen et al., Mol. Gen Genet. 177: 65 (1979)), and the 3′ regionfrom transcript 7 of pTiA6 (Currier and Nester, J. Bact. 126: 157-165(1976)). The vector is constructed in a multi-step process detailedbelow.

The plasmid pCGN783 is deposited with ATCC, accession number 67868.

A. Construction of pCGN739

To obtain the gentamnicin resistance marker, the resistance gene isisolated from a 3.1 kb EcoRI-PstI fragment of pPhIJI (Hirsch et al.1984, supra) and cloned into pUC9 (Vieira and Messing, Gene 19: 259-268(1982)), yielding pCGN549. The pCGN549 HindIII-BamHI fragment containingthe gentamicin resistance gene replaces the HindIII-BglII fragment ofpCGN587 (for above) constructing pCGN594. The pCGN594 HindIII-BamHIregion which contains an ocs-kanamnycin-ocs fragment is replaced withthe HindIII-BamHI polylinker region from pUC18 (Yanisch-Perron et al.,Gene 53: 103-119 (1985)) to make pCGN739.

B. Construction of pCGN726C

pCGN566 contains the EcoRI-HindIII linker of pUC18 Yanisch-Perron etal., Gene 53: 103-119 (1985), inserted into the EcoRI-HindHIII sites ofpUC13-cm (K. Buckley, Ph.D. Thesis, UC San Diego 1985). TheHindIII-BglII fragment of pNW31c-8, 29-1 (Thomashow et al., Cell 19:729-739 (1980)) containing ORF1 and 2 (Barker et al., Plant Mol. Biol.2: 335-350 (1983)) is subcloned into the HindII-BamHI sites of pCGN566producing pCGN703. The Sau3A fragment of pCGN703 containing the 3′region of transcript 7 from pTiA6 (corresponding to bases 2396-2920 ofpTi15955; Barker et al., Plant Mo. Biol. 2:335-350 (1983)) is subclonedinto the BamHI site of pUC18 (Yanisch-Perron al., Gene 53:103-119(1985)) producing pCGN709.

The EcoRI-SmaI polylinker region of pCGN709 is replaced with theEcoRI-SmaI fragment from pCGN587 (for production see infra) whichcontains the kanamycin resistance gene (APH3 'II) producing pCGN726.

The EcoRI-SalI fragment of pCGN726 plus the BglII-EcoRI fragment ofpCGN734 are inserted into the BamHI-SalI sites of pUC8-pUC13-cm(chloramphenical resistant, K. Buckley, PH.D. Thesis, UC San Diego 1985)producing pCGN738. To construct pCGN734, the HindIII-SphI fragment ofpTiA6 corresponding to bases 3390-3241 (Barker et al., Plant Mo. Biol.2:335-350 (1983)) is cloned into the HindIII-SphI site of M13mp19(Yanisch-Perron et al., Gene 53: 103-119 (1985); Norrander et al. Gene26: 101-106 (1983)). Using an oligonucleotide corresponding to bases3287 to 3300, DNA synthesis is primed from this template. Following S1nuclease treatment and HindIII digestion, the resulting fragment iscloned in the HindIII-SmaI site of pUC19 (Yanisch-Perron et al., Gene53: 103-119 (1985)). The resulting EcoRI-HindIII fragment correspondingto bases 3287-3390 (Barker et al., Plant Mo. Biol. 2: 335-350 (1983)),is cloned with the EcoRI-HindHIII fragment of pTiA6 (corresponding tobases 3390-4494) into the EcoRI site of pUC8 (Vieira and Messing, Gene19: 259-268 (1982)) resulting in pCGN374. pCGN726c is derived frompCGN738 by deleting the 900bp EcoRI-EcoRI fragment.

C. Construction of pCGN766C

The HindIII-BamHI fragment of pCGN167 (see infra) containing theCAMV-35S promoter, 1 ATG-kanamycin gene and the BamHI fragment 19 ofpTiA6 is cloned into the BamHI-HindIII sites of pUC19 (Norrander et al.Gene 26:101-106 (1983); Yanisch-Perron et al., Gene 53: 103-119 (1985))constructing pCGN976.

The 35S promoter and 3′ region from transcript 7 is developed byinserting a 0.7 kb HindIII-EcoRI fragment of pCGN976 (35S promoter) andthe 0.5 kB EcoRI-SalI fragment of PCGN709 (transcript 7:3′) into theHindIII-SalI sites of pCGN566 constructing pCGN766c. To constructpCGN167, the AluI fragment of CAMV (bp 7144-7735) (Gardner et al., Nucl.Acids Res. 9: 2871-2888 (1981)) is obtained by digestion with AluI andcloned into the HindII site of M13mp7 (Vieira and Messing, Gene 19:259-268 (1982) to create C614. An EcoRI digest of C614 produces theEcoRI fragment from C614 containing the 35S promoter which is clonedinto the ECoRI site of pIC8 (Vieira and Messing, Gene 19: 259-268(1982)) to produce pCGN146.

To trim the promoter region, the Bell site (bp 7670) is treated withBglII and Bal31 and subsequently a BglII linker is attached to the Bal31treated DNA to produce pCGN147. pCGN148a containing a promoter region,selectable marker (KAN with 2 ATG's) and 3′ region is prepared bydigesting pCGN528 (see below) with BglII and inserting the BamHI-BglIIpromoter fragment from pCGN147. This fragment is cloned into the BglIIsite of pCGN528 so that the GglII site is proximal to the kanamycin geneof pCGN528.

The shuttle vector used for this construct, pCGN528, is made as follows.pCGN525 is made by digesting a plasmid containing Tn5 which harbors akanamycin gene (Jorgenson et al., Mol. Gen. Genet. 177: 565 (1979)) withHindIII-BamHI and inserting the HindIII-BamHI fragment containing thekanamycin gene into the HindIII-BamHI sites in the tetracycline gene ofpACYC184 (Chang and Cohen, J. Bacteriol. 134: 1141-1156 (1978)). pCGN526is made by inserting the BamHI fragment 19 of pTiA6 (Thomashow et al.,Cell 19:729-739 (1980)) into the BamHI site of pCGN525. pCGN528 isobtained by deleting the small XhoI fragment from pCGN526 by digestingwith XhoI and relegating.

pCGN149a is made by cloning the BamHI kanamycin gene fragment from9MB9KanXXI into the BamHI site of pCGN148a.

pMB9KanXXI is a pUC4k variant (Vieira and Messing, Gene 19:259-268(1982)) which has the XhoI site missing but containing a functionalkanamycin gene from Tn903 to allow for efficient selection inAgrobacterium.

pCGN149a is digested with BglII and SphI. This small BglII-SphI fragmentof pCGN149A is replaced with the BamHII-SphI fragment from MI (seebelow) isolated by digestion with BamHI and SphI. This produces pCGN167,a construct containing a full length CaMV promoter, 1ATG-Kanamycin gene,3′ end and the bacterial Tn903-type kanamycin gene. MI is an EcoRIfragment from pCGN550 (see construction of pCGN587) and is cloned intothe EcoRI cloning site of M13mp9 in such a way that the PstI site in the1ATG-kanamycin gene is proximal to the polylinker region of M13mp9.

D. Construction of pCGN451

pCGN451 contains the ocs5′-ocs3′ Cassette cloned into a derivative ofpUC8 (Vieira and Messing, Gene 19: 259-268 (1982)). The modified vectoris derived by digesting pUC8 with HincII and ligating in the presencesynthetic linker DNA, creating pCGN416, and then deleting the EcoRI siteof pCGN416 by EcoRI digestion followed by treatment with Klenow enzymeand self ligation to create pCGN426.

The ocs5′-ocs3′ Cassette is constructed by a series of steps from DNAderived from the octopine Ti-plasmid pTiA6 (Currier and Nester, J. Bact.126: 157-165 (1976)). An EcoRI fragment of pTiA6 (bp 13362-16202; thenumbering is by Barker et al., Plant Mol. Biol. 2: 335-350 (1983), forthe closely related Ti plasmid pTi 15955) is removed from pVK232 (Knaufand Nester, Plasmid 8: 45 (1982)) by EcoRI digestion and cloned intoEcoRI digested pACYC184 (Chang and Cohen, J. Bacteriol. 134: 1141-1156(1978)) to generate pCGN15. The 2.4 kb BamHI-EcoRI fragment (bp13774-16202) of pCGN15 is cloned into EcoRI-BamHI digested pBR322(Bolivar et al., Gene 2: 95-113 (1977)) to yield pCGN429. The 412 bpEcoRI-BamHI fragment (bp13362-13774) of pCGN15 is cloned intoEcoRI-BamHI digested pBR3322 (Bolivar et al., Gene 2: 95-113 (1977)) toyield pCGN407. The cut-down promoter fragment is obtained by digestingpCGN407 with XmnI (bp 13512), followed by resection with Bal31exonuclease, ligation of synthetic EcoRI linkers, and digestion withBamHI. Resulting fragments of approximately 130 bp are gel purified andcloned into M13mp9 (Vieira and Messing, Gene 19: 259-268 (1982)) andsequenced. A clone, I-4, in which the EcoRI linker had been inserted atbp 13642 between the transcription initiation point and the translationinitiation codon is identified by comparison with the sequence of deGreve et al., J. Mol. Appl. Genet. 1: 499-512 (1982)); the EcoRIcleavage site is at position 13639, downstream from the mRNA start site.The 141 bp EcoRI-BamHI fragment of I-4, containing the cut-downpromoter, is cloned into EcoRI-BamHI digested pBR322 (Bolivar et al.,Gene 2: 95-113 (1977)) to create pCGN428. The 141 bp EcoRI-BamHIpromoter piece from pCGN428, and the 2.5 kb EcoRI-BamHI ocs 5′ piecefrom pCGN429 are cloned together into EcoRI digested pUC9 (Vicira andMessing, Gene 19: 259-268 (1982)) to generate pCGN442, reconstructingthe ocs upstream region with a cut-down promoter section.

The HindIII fragment of pLB41 (D. Figurski) containing the gentamycinresistance gene is cloned into HindIII digested pACYC184 (Chang andCohen, J. Bacterol. 134: 1141-1156 (1978)) to create pcDNA413b. The 4.7kb BamHI fragment of pTiA6 (Currier and Nester, J. Bact. 126: 157-165(1976)) containing the osc 3′ region, is cloned into BamHI digestedpBR325 (F. Bolivar, Gene 4: 121-136 (1978)) to create 33c-19. The SmaIsite a position 11207 of 33c-19 is converted to an XhoI suite usingsynthetic XhoI linker DNA, generating pCGN401.2. The 3.8 kb BamHI-EcoRIfragment of pCGN401.2 is cloned into BamHI-EcoRI digested pCGN413b tocreate pCGN419.

The ocs5′-osc3′ Cassette is generated by cloning the 2.64 kb EcoRIfragment of pCGN442, containing the 5′ region, into EcoRI digestedpCGN419 to create pCGN446. The 3.1 kb XhoI fragment of pCGN446, havingthe ocs 5′ region (bp13639-15208) and ocs 3′ region (bp 11207-12823), iscloned into the XhoI site of pCGN426 to create pCGN451.

E. Construction of pCGN587

The HindIII-SmaI fragment of Tn5 containing the entire structural genefor APH3′II (Jorgensen et al., Mol. Gen. Genet. 177: 65 (1979)) iscloned into pUC8 (Vieira and Messing, Gene 12: 259-268 (1982)) thisconverts the fragment into HindIII-EcoRI fragment, since there is anEcoRI site immediately adjacent to the SmaI site. The PstI-EcoRIfragment of pCGN300, containing the 3′-portion of the APH3′II gene, isthen combined with an EcoRI-BamHI-SalI-PstI linker into the EcoRI siteof pUC7 to make pCGN546W]. An ATG codon is upstream from and out ofreading frame with the ATG initiation codon of APH3′II. The undesiredATG is avoided by inserting a Sau3A-PstI fragment from the 5′-end ofAPH3′II, which fragment lacks the superfluous ATG, into the BamHI-PstIsite of pCGN546W to provide plasmid pCGN550. The EcoRI fragment ofpCGN550 containing the APH3′II gene is then cloned into the EcoRI siteof pUS8-pIC13-cm (K. Buckley (1985), supra) to give pCGN551. The plasmidpCGN451 (described above) having the ocs 5′ and the ocs 3′ in the properorientation is digested with EcoRI and the EcoRI fragment from pCGN551containing the intact kanamycin resistance gene is inserted into theEcoRI site to provide pCGN552 having the kanamycin resistance gene inthe proper orientation.

This ocs/KAN gene is used to provide a selectable marker for the transtype binary victor pCGN587.

The 5′ portion of the engineered octopine synthase promoter cassetteconsists of pTiA6 DNA from the XhoI at bp 15208-13644 (Barker et al.,Plant Mo. Biol. 2: 335-350 (1983)), which also contains the T-DNAboundary sequence (border) implicated in T-DNA transfer. In the plasmidpCGN587, the ocs/KAN gene form pCGN552 provides a selectable marker aswell the right border. The left boundary region is first cloned inM13mp9 as a HindIII-SmaI piece (pCGN502) (base pairs 602-2212) andrecloned as a KpnI-EcoRI fragment in pCGN565 to provide pCGN580. pCGN565is a cloning vector based on pUC-pUC13-Cm, (K. Buckley, Ph.D. Thesis, UCSan Diego 1985 but containing pUC18 linkers; Yanisch-Perron et al., Gene53:103-119 (1985)) pCGN580 is linearized with BamHI and used to replacethe smaller BglI fragment of pVCK102 (Knauf and Nester, Plasmid 8: 45(1982)), creating pCGN585. By replacing the smaller SalI fragment of pCGN585 with the XhoI fragment from pCGN552 containing the ocs/KAN gene,pCGN587 is obtained.

E. Final Construction of pCGN783

The 0.7 kb HindIII-EcoRI fragment of pCGN766 (CaMV-35S promoter) isligated to the 1.5 kb EcoRI-SalI fragment of pCGN726c (2 ATG3-KAN-3′region) into the HindIII-SalI sites of pUC119 (J. Vieira, RutgersUniversity, N.J.) to produce pCGN778. The 2.2 kb region of pCGN778,HindIII-SalI fragment containing the CaMV 35S promoter (1-ATG-KAN-3′region) replaces the HindHIII-SalI polylinker region of pCGN739 toproduce pCGN783.

Example 84 Construction of pCGN1539 and pCGN1540

CGN1539 and pCGN1540 are binary plant transformation vectors containingthe left and right T-DNA borders of Agrobacterium tumefaciens octopineTi-plasmid pTiA6 (Currier and Nester, J. Bact. 126: 157-165 (1976)), thegentamycin resistance gene of pPHiJI (Hirsch and Beringer, Plasmid 12:139-141 (1984)), an agrobacterium rhizogenes Ri plasmid origin ofreplication from pLJB11 (Jouanin et al., Mol. Gen. Genet. 201: 370-374(1985)), the mas promoter region and mas 3′ region of pTiA6 with thekanamycin resistance gene of Tn5 (Jorgensen et al., Mol. Gen. Genet.177: 65 (1979)) a ColE1 origin of replication from pBR322 (Bolivar etal., Gene 2: 95-113 (1977)), and a lacZ′ screenable marker gene frompUC18 (Norrander et al., Gene 26: 101-106 (1983)). The backbone ofpCGN1539-1540, containing the gentamycin resistance gene and the Ri andColE1 origins, is derived from pCGN1532 (see below). The Ti borders andplant selectable marker gene (mas 5′-kan-mas3′), are from pCGN1537; theplant selectable marker cassette is in turn taken from pCGN1536, whilethe right border and the lacZ′ fragments are derived from pCGN565RBx2X,and the left border is derived from pCGN65.

A. pCGN1532 Construction

The 3.5 kb EcoRI-PstI fragment containing the gentamycin resistance geneis removed from pPhlJI (Hirsch and Beringer, Plasmid 12: 139-141 (1984))by EcoRI-PstI digestion and cloned into EcoRI-PstI digested pUC9 (Vieiraand Messing, Gene 19: 259-268 (1982)) to generate pCGN549. HindHIII-PstIdigestion of pCGN549 yields a 3.1 kb fragment bearing the gentamycinresistance gene, which is made blunt ended by the Klenow fragment of DNApolymerase I and cloned into PvuII digested pBR322 (Bolivar et al., Gene2: 95-113 (1977) to create pBR322GM. pBR322Gm is digested with DraI andSphI, treated with Klenow enzyme to create blunt ends, and the 2.8 kbfragment cloned into the Ri origin containing plasmid pLJbB11 (Jouaninet al., ol. Gen. Genet. 201: 370-374 (1985)) which had been digestedwith ApaI and made blunt ended with Klenow enzyme, creating pLHbB11Gm.The extra ColE1 origin and the kanamycin resistance gene are deletedfrom pLJvB11GM by digestion with BaHI followed by self closure to createpGMB11. The HindII site of pGmB11 is deleted by HindII digestionfollowed by treatment with Klenow enzyme and self closure, creatingpGmB11-H. The PstI site of pGmB11-H is deleted by PstI digestionfollowed by treatment with Klenow enzyme and self closure, creatingpCGN1532.

B. pCGN1536 Construction

The 5.4 kb EcoRI fragment is removed from pVK232 (Knauf and Nester,Plasmid 8: 45 (1982)) by EcoRI digestion and cloned into EcoRI digestedpACYC184 (Chang and Cohen, J. Bacteriol. 134: 1141-1156 (1978)) tocreate pCGN14. The 1434 bp ClaI-SphI fragment of pCGN14, containing themas 5′ region (bp20128-21562 according to numbering of (Barker et al.,Plant Mo. Biol. 2: 335-350 (1983)) is cloned into AccI-SphI digestedpUC19 (Yanisch-Perron et al., Gene 53: 103-119 (1985)) to generatepCGN50. A 746 bp EcoRV-NaeI fragment of the mas 5′ region is replaced byan XhoI site by digesting pCGN40 with EcoRV and NaeI followed byligation in the presence of a synthetic XhoI linker DNA to createpCGN1036. The 765 bp SstI-HindHIII fragment (bp 18474-19239) of pCGN14,containing the mas 3′ region, is cloned into SstI-HindHIII digestedpUC18 (Norrander et al., Gene 26: 101-106 (1983)) to yield pCGN43. TheHindIII site of pCGN43 is replaced with an EcoRI site by digestion withHindIII, blunt ending with Klenow enzymne, and ligation of syntheticEcoRI linker DNA to create pCGN1034.

The 767 bp EcoRI fragment of pCGN 1034 is cloned into EcoRI-digestedpCGN1036 in the orientation that placed bp 19239 of the mas 3′ regionproximal to the mas 5′ region to create pCGN1040. pCGN1040 is subjectedto partial digestion with SstI, treated with T4 DNA polymerase to createblunt ends, and ligated in the presence of synthetic XhoI linker DNA; aclone is selected in which only the SstI site at the junction of bp18474 and vector DNA (constructed in pCGN43 and carried into pCGN1040)is replaced by an XhoI site to generate pCGN1047.

pCGN565 (see above) is digested with EcoRI and HindIII, treated withKlenow enzyme to create blunt ends, and ligated in the presence ofsynthetic XhoI linker DNA to create pCGN1003; this recreates the EcoRIsite adjacent to the XhoI linker. pCGN1003 is digested with EcoRI,treated with Klenow enzyme to create blunt ends, and ligated in thepresence of synthetic PstI linker DNA to create pCGN1007. The 1.5 kbXhoI fragment of pCGN1047, containing the mas 5′ region and the mas 3′region with a multiple cloning site between, is cloned into XhoIdigested pCGN1007 to construct pCGN1052. A portion of the multiplecloning site of pCGN1052 is deleted by digestion with XbaI and SstI,treated with Klenow enzyme to make blunt ends, and ligated to generatepCGN1052deltaXS.

The 1 kb EcoRI-SmaI fragment of pCGN550 (pCGN783 description),containing the 1 ATG-kanamycin resistance gene, is cloned intoEcoRI-SmaI digested Bluescript M13-KS (Strategene, Inc.) to createpBSKm; this plasmid contains an M13 region. allowing generation ofsingle stranded DNA. Single stranded DNA is generated according to thesupplier's recommendations, and in vitro mutagenesis is performed(Adelman et al., DNA 2: 183-193 (1983)) using a syntheticoligonucleotide with the sequence 5′GAACTCCAGGACGAGGC3′ (SEQ ID No. 97)to alter a PstI site within the kanamycin resistance gene and make itundigestable, creating pCGN1534. pCGN1534 is digested with SmaI andligated in the presence of synthetic EcoRI linker DNA to generatepCGN1535.

The 1 kb EcoRI fragment of pCGN1536 is cloned into EcoRI digestedpCGN1052deltaXS to create the mas5′-kan mas3′ plant selectable markercassette pCGN1536.

C. pCGN565RAx2X Construction

pCGN451 (pCGN783 description) is digested with HpaI and ligated in thepresence of synthetic SphI linker DNA to generate pCGN55. The XhoI-SphIfragment of pCGN55 (bp13800-15208, including the right border, ofAgrobacterium tumefaciens T-DNA; (Barker et al., Gene 2: 95-113 (1977))is cloned into SalI-SphI digested pUC19 (Yanisch-Perron et al., Gene 53:103-119 (1985)) to create pCGN60. The 1.4 kb HindIII-BamHI fragment ofpCGN60 is cloned into HindIII-BamHi digested pSP64 (Promega, Inc.) togenerate pCGN1039. pCGN1039 is digested with SmaI and NruI (deletingbp14273-15208; Barker et al., Gene 2: 95-113 (1977)) and ligated in thepresence of synthetic BglII linker DNA creating pCGN1039deltaNS The 0.47kb EcoRI-HindIII fragment of pCGN1039deltaNS is cloned intoEco-RI-HindIII digested pCGN565 (described in pCFN783 description) tocreate pCGN565RB. The HindIII site of pCGN565RB is replaced with an XhoIsite by HindIII digestion, treatment with Klenow enzyme, and ligation inthe presence of synthetic XhoI linker DNA to create pCGN565RB-H+X.

pUC18 (Norrander et al. Gene 26: 101-106 (1983) is digested with HaeIIto release the lacZ′ fragment, treated with Klenow enzyme to createblunt ends, and the lacZ′-containing fragment ligated intopCGN56SRB-H+X, which had been digested with AccI and SphI and treatedwith Klenow enzyme, in such an orientation that the lacZ′ promoter isproximal to the right border fragment; this construct, pCGN565RBx2x ispositive for lacZ′ expression when plated on an appropriate host andcontains bp 13990-14273 of the right border fragment (Barker et al.,Plant Mo Biol. 2: 335-350 (1983)) having deleted the AccI-SphI fragment(bp 13800-13990).

D. pCGN65 Construction

pCGN501 is constructed by cloning a 1.85 kb EcoRI-XhoI fragment of pTiA6(Currier and Nester, J. Bact. 126: 157-165 (1976)) containing bases13362-15208 (Barker et al., Plant Mol. Biol. 2: 335-350 (1983)) of theT-DNA (right border), into EcoRI-SalI digested M13mp9 (Vieira andMessing, Gene (1982) 19: 259-268 (1982). PCGN502 is constructed bycloning a 1.6 kb HindIII-SmaI fragment of pTiA6, containing bases602-2212 of the T-DNA (left :border), into HindIII-SmaI digested M13mp9.pCGN501 and pCGN502 are both digested with EcoRI and HindIII and bothT-DNA-containing fragments cloned together into HindIII digested pUC9(Vieira and Messing, Gene 19: 259-268 (1982)) to yield pCGN503,containing both T-DNA border fragments. pCGN503 is digested with HindIIIand EcoRI and the two resulting HindIII-EcoRI fragments (containing theT-DNA borders) are cloned into EcoRI digested pHC79 (Hohn and Collins,Gene 11: 291-298 (1980)) to generate pCGN518. The KpnI-EcoRI fragmentfrom pCGN518, containing the left T-DNA border, is cloned intoKpnI-EcoRI digested pCGN565 to generated pCGN580. The BamHI-BglIIfragment of pCGN580 is cloned into the BamHI site of pACYC184 (Chang andCohen, J. Bacteriol. 134: 1141-1156 (1978)) to create pCGN51. The 1.4 kbBamHI-SphI fragment of pCGN60 (see pCGN65x2X section above) containingthe T-DNA right border fragment, is cloned into BamHI-SphI digestedpCGN51 to create pCGN65.

E. pCGN1537 Construction

pCGN65 is digested with KpnI and XbaI, treated with Klenow enzyme tocreate blunt ends, and ligated in the presence of synthetic BglII linkerDNA to create pCGN65deltaKX. pCGN65deltaKX is digested with SalI,treated with Klenow enzyme to create blunt ends, and ligated in thepresence of synthetic XhoI linker DNA to create pCGN65deltaKX-S+X. The728 bp BglII-XhoI fragment of pCGNRBx2X, containing the T-DNA rightborder piece and the lacZ′ gene, is cloned into BglII-XhoI digestedpCGN65deltaKX-S+X, replacing pCGN65x2X. The ClaI fragment pCGN65x2X isdeleted and replaced with an XhoI linker by digesting with ClaI,treating with Klenow enzyme to create blunt ends, and ligating in thepresence of synthetic XhoI linker DNA to create pCGN65delta2XX.

pCGN65delta2XX is digested with BglII and fused with BglII digestedpCGN549 (see pCGN1532 section above) to create pCGN1530 which containedboth plasmid backbones. pCGN1530 is digested with XhoI and religated,then a gentamycin-resistant cholramphenicol-sensitive clone is chosenwhich had deleted the pACYC184-derived backbone, creating pCGN1530A. The2.43 kb XhoI fragment of pCGN1536, containing the mas5′-kan-mas3′cassette, is cloned into XhoI digested pCGN1530A to create pCGN1537.

F. Final Assembly of pCGN1540

The BglII fragment of pCGN1537, containing the plant selectable markergene and the lacZ′ screenable marker gene (with multiple cloning site),all between the T-DNA borders, is cloned into BamHI digested pCGN1532. Aclone of the orientation bearing the T-DNA right border adjacent to thepBR322 origin of replication is designated pCGN1539, and the orientationbearing the T-DNA right border adjacent to the Ri plasmid origin ofreplication is designated pCGN1540. This binary vectors has severaladvantageous features, including a minimal amount of DNA between theT-DNA borders, high stability in Agrobacterium hosts, high copy numberin E. coli hosts, and a blue/white screen with multiple restrictionenzyme sites for ease of cloning target DNA.

The plasmid pCGN1540 has been deposited with ATCC, accession number40586.

2. Binary Vectors Containing Chimeric Genes

This section details the subcloning of the expression cassette into thebinary vector. In the previous section the construction of pCGN783 andpCGN1540 are detailed. These are binary vectors which can be used inAgrobacterium mediated tansformation experiments to transform plants.The vectors are designed to cotransform a chimeric gene of interest intoa plant. However, the chimeric gene first must be subcloned into thebinary vector. The following section details the subcloning of thechimeric genes constructed in Section 6 into either the pCGN783 or thepCGN1540 binary vectors. The resulting vectors are capable oftransforming plants with the chimeric gene.

Example 85 Construction of pCGN1754 and pCGN1760 (pCGN783 Containing theEmpty SSU Promoter Cassette in Either Orintation)

The BamHI site of pCGN783 lies near the right T-DNA border, with theplant selectable marker gene lying between the left T-DNA border and theBamHI site. Cloning a chimeric gene construct into the BamHI site placesthe chimeric gene between the plant selectable marker gene and the rightT-DNA border. The unique BglII site of pCGN1509 lies in a non-essentialportion of the ocs 3′ region.

pCGN1509 is digested with BglII and the entire vector is cloned into theBamHI site of pCGN783, and both possible orientations are recovered. Aplasmid in which the RuBISCO small subunit promoter and ocs 3′ regionsare proximal to the right T-DNA border of pCGN783 is designatedpCGN1754, and a plasmid in which the RuBISCO small subunit promoter andocs 3′ regions are proximal to the plant selectable marker gene ofpCGN783 is designated pCGN1760.

Example 86 Construction of pCGN1755A, pCGN1755B, pCGN1755C, andpCGN1755D (pCGN783 Containing the SSU/PR-1a (Sense and Anti-Sense)Expression Cassette in Either Orientation)

pCGN1752A is digested with BglII and the entire vector is cloned intoBamHI digested pCGN783, and both possible orientations are recovered. Aplasmid in which the RuBISCO small subunit promoter-PR1-ocs 3′ regionsare proximal to the right T-DNA border of pCGN783 is designatedpCGN1755C, and a plasmid in which the RuBISCO small subunitpromoter-PR1-ocs 3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1755A.

pCGN1752B is digested with BglII and the entire vector is cloned intoBamHI digested pCGN783, and both possible orientations are recovered. Aplasmid in which the RUBISCO small subunit promoter-PR1-ocs 3′ regionsare proximal to the right T-DNA border of pCGN783 is designatedpCGN1755B, and a plasmid in which the RuBISCO small subunitpromoter-PR1-ocs 3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1755B.

Example 87 Construction of pCGN1756A, pCGN1756B, pCGN1756C, andpCGN1756D (pCGN783 Containing the SSU Promoter/PR-1b (Sense andAnti-Sense) Expression Cassette in Either Orientation)

pCGN1753A is digested with BglII and the entire vector is cloned intoBamHI digested pCGN783, and both possible orientations are recovered. Aplasmid in which the RuBISCO small subunit promoter-PR1-ocs 3′ regionsare proximal to the right T-DNA border of pCGN783 is designatedpCGN1756C, and a plasmid in which the RuBISCO small subunitpromoter-PR1-ocs 3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1756A.

pCTN1753B is digested with the BglII and cloned into BamHI digestedpCGN783, and both possible orientations are recovered. A plasmid inwhich the RuBISCO small subunit promoter-PR1-ocs 3′ regions are proximalto the right T-DNA border of pCGN783 is designated pCGN1756D, and aplasmid in which the RuBISCO small subunit promoter-PR1-ocs 3 regionsare proximal to the plant selectable marker gene pCGN783 is designatedpCGN1756B.

Example 88 Construction of PCGN1766 and pCGN1767 (pCGN783 Containing anEmpty Double CAMV 35S Promoter Cassette in Either Orientation)

pCGN1761 is digested with BamHI and the entire vector is cloned intoBamHI digested pCGN783, and both possible orientations are recovered. Aplasmid in which the double CAMV 35S promoter and tml 3′ regions areproximal to the plant selectable marker gene of pCGN783 is designatedpCGN1767, and a plasmid in which the double 35S promoter and tml 3′regions are proximal to the right T-DNA border of pCGN783 is designatedpCGN1766.

Example 89 Construction of pCGN1764A, pCGN1764B, pCGN1764C, andpCGN1764D (Double CAMV 35S Promoter/PR1a (Sense and Anti-Sense) intopCGN783)

pCGN1762A is digested with BamHI and cloned into BamHI digested pCGN783,and both possible orientations are recovered. A plasmid in which thedouble 35S-PR1-tml3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1764A, and a clone in which the double35S-PR1-tml3′ regions are proximal to the right T-DNA border of pCGN783is designated pCGN1764C.

pCGN1762B is digested with BamHI and cloned into BamHI digested pCGN783,and both orientations are recovered. A plasmid in which the double 35Spromoter is proximal to the plant selectable marker is designatedpCGN1764B. A plasmid in the opposite orientation is designatedpCGN1764D.

Example 90 Construction of pCGN1765A, pCGN1765B, pCGN1765C, andpCGN1765D (Double CAMV 35S Promoter/PR-1b (Sense and Anti-Sense) intopCGN783 in Either Orientation)

pCGN1763A is digested with BamHI and cloned into BamHI digested pCGN783,and both possible orientations ame recovered. A plasmid in which thedouble 35S-PR1-tml3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1765A, and a plasmid in which thedouble35S-PR1-tml3′ regions are proximal to the right T-DNA border ofpCGN783 is designated pCGN1765C.

pCGN1763B is digested with BamHI and cloned into BamHI digested pCGN783and both possible orientations are recovered. A plasmid in which thedouble 35S-PR1-tml3′ regions are proximal to the plant selectable markergene of pCGN783 is designated pCGN1765B, and a plasmid in which thedouble35S-PR1-tml3′ regions are proximal to the right T-DNA border ofpCGN783 is designated pCGN1765D.

Example 91 Construction of pCGN1780A, pCGN1780B, pCGN1780C, pCGN1780D(Double CAMV35S Promoter/Cucumber Chitinase/Lysozyme (Sense andAnti-Sense) into pCGN783 in Either Orientation)

pCIB1000 is digested with BamHI and cloned into BamHI site in pCGN783,and both possible orientations are recovered. A plasmid in which thedouble 35S-chitinase-tml3′ regions are proximal to the plant selectablemarker gene of pCGN783 is designated pCGN1780A, and a plasmid in whichthe double 35S-chitinase-tml3′ regions are proximal to the right T-DNAborder of pCGN783 is designated pCGN1780C.

pCIB1001 is digested with BamHI and cloned into BamHI digested pCGN783in either orientation. A plasmid in which the double 35S-chitinase-tml3′regions are proximal to the plant selectable marker gene of pCGN783 isdesignated pCGN1780B. A plasmid in which the double 35S-chitinase-tml3′regions are proximal to right T-DNA border of pCGN783 is designatedpCGN1780D.

Example 92 Construction of pCGN1789 (Double CAMV 35S Promoter EmptyCassette into pCGN1540 in Either Orientation)

The 2.36 kb XbaI-PstI fragment of pCGN1431 is subcloned into XbaI-PstIdigested pCGN1540 to create the plasmid pCGN1789. This plasmid has theinsert oriented in a direction such that the double 35S CAMV promoter isproximal to the plant selectable marker.

Example 93 Construction of pCGN1774A, pCGN1774B, pCGN1774C, andpCGN1774D (Double CAMV 35S Promoter/PR-1a (Sense and Anti-Sense) intopCGN1540 in Either Orientation)

The 4.2 kb XbaI fragment of pCGN1762A is subcloned into XbaI digestedpCGN1540, and both possible orientations are recovered. A plasmid inwhich the double 35S promoter fragment is proximal to the right T-DNAborder of pCGN1540 is designated pCGN1774A, and a plasmid in which thedouble 35S promoter fragment is proximal to the plant selectable markergene of pCGN1540 is designated pCGN1774C.

The 42 kb XbaI fragment of pCGN1762B is cloned into XbaI digestedpCGN1540, and both possible orientations are recovered. A plasmid inwhich the double 35S promoter fragment is proximal to the right T-DNAborder of pCGN1540 is designated pCGN1774B, and a plasmid in which thedouble 35S promoter fragment is proximal to the plant selectable markergene of pCGN1540 is designated pCGN1774D.

Example 94 Construction of pCGN1775A, pCGN1775B, pCGN1775C, andpCGN1775D (Double CAMV 35S Promoter/PR-1b (Sense and Anti-Sense) intopCGN1540 in Either Orientaton)

The 4.1 kb XbaI fragment of pCGN1763A is cloned into XbaI digestedpCGN1540S, and both possible orientations are recovered. A plasmid inwhich the double 35S promoter fragment is proximal to the right T-DNAborder of pCGN1540 is designated pCGN1775A, and a plasmid in which thedouble 35S promoter fragment is proximal to the plant selectable markergene of pCGN1540 is designated pCGN1775C.

The 4.1 kb XbaI fragment of pCGN1763B is cloned into XbaI digestedpCGN1540, and both possible orientations are recovered. A plasmid inwhich the double 35S promoter fragment is proximal to the right T-DNAboiler of pCGN1540 is designated pCGN1775B, and a clone in which thedouble 35S promoter fragment is proximal to the plant selectable markergene of pCGN1540 is designated pCGN1775D.

Example 95 Construction of pCGN1783C and pCGN1783D) (Double CAMV 35SPromoter/PR-R major (Sense and Anti-Sense) into pCGN1540 in EitherOrientation)

The 4.5 kb XbaI fragment of pCIB1002 is subcloned into XbaI digestedpCBN1540 in such an orientation that the double 35S promoter is proximalto the plant selectable marker gene of 1540. This plasmid is designatedas pCN1783C.

The 4.5 kb XbaI fragment of pCIB1003 is subcloned into XbaI digestedpCGN1540 in such an orientation that the double 35S promoter fragment isproximal to the plant selectable marker gene of pCGN1540 to createpCGN1783D.

Example 96 Construction of pCIB1026 and pCIB1027 (Double CAMV 35SPromoter/PRP (Sense and Anti-Sense) into pCGN1540 in Either Orientation)

The XbaI fragment of pCIB1020, which contains the chimeric PR-P gene, issubcloned into XbaI digested pCGN1540 in such an orientation that thepromoter fragment is proximal to the plant selectable marker gene. Thisplasmid is designated as pCIB1026.

The XbaI fragment of pCIB1021, which contains the chimeric PR-Panti-sense gene, is subcloned into XbaI digested pCGN1540 in such anorientation that the promoter fragment is proximal to the plantselectable marker gene. This plasmid is designated as pCIB1027.

Example 97 Construction of pCGN1791C and pCGN1791D (Double CAMV 35SPromoter/PR-Q (Sense and Anti-Sense) into pCGN1540 in EitherOrientation)

The XbaI fragment from pCIB1022, which contains the chimeric PR-Q gene,is subcloned into XbaI digested pCGN1540 in an orientation such that thepromoter fragment is proximal to the plant selectable marker. Thisplasmid is designated as pCGN1791C.

The XbaI fragment from pCIB1023, which contains the chimeric PR-Qanti-sense gene, is subcloned into XbaI digested pCGN1540 in such anorientation that the promoter fragment is proximal to the plantselectable marker. The plasmid is designated pCGN1791D.

Example 98 Construction of pCIB1030 and pCIB1031 (Double CAMV 35SPromoter/PR-O′ (Sense and Ani-Sense) into pCGN1540 in EitherOrientation)

The XbaI fragment of pCIB1024, which contains the chimeric PR-O′ gene issubcloned into XbaI digested pCGN1540 in such a way that the 35 Spromoter is proximal to the plant selectable marker. The plasmid isdesignated pCIB1030.

The XbaI fragment of pCIB1025, which contains the chimeric PR-O′anti-sense gene, is subcloned into XbaI digested pCGN1540 in a similarorientation as pCIB1030. The new plasmid is designated pCIB1031.

Example 99 Construction of pCIB1030A and pCIB1031B (double CAMV 35SPromoter/PR-O′ (Sense and Anti-Sense) into pCGN1540)

The XbaI fragment of pCIB1024A, which contains the chimeric PR-O′ gene,is subcloned into XbaI digested pCGN1540 in such a way that the 35Spromoter is proximal to the plant selectable marker. This plasmid isdesignated as pCIB1030A.

The XbaI fragment of pCIB1025A, which contains the chimeric PR′ gene inan anti-sense orientation, is subcloned into XbaI digested pCGN1540 insuch a way that the 35S promoter is proximal to the plant selectablemarker. The resulting plasmid is designated pCIB1031A.

Example 100 Construction of pCIB1042 and pCIB1043 (Double CAMV 35SPromoter/PR-2 (Sense and Anti-Sense) into pCGN1540)

The XbaI fragment of pCIB1032, which contains the chimeric PR-2 gene, issubcloned into XbaI digested pCGN1540 in such a way that the 35Spromoter is proximal to the plant selectable marker. This plasmid isdesignated as pCIB1042.

The XbaI fragment of pCIB1033, which contains the chimeric PR-2 gene inan anti-sense orientation, is subcloned into XbaI digested pCGN1540 insuch a way that the 35S promoter is proximal to the plant selectablemarker. The resulting plasmid is designated pCIB1043.

Example 101 Construction of pCIB1044 and pCIB1045 (Double CAMV 35SPromoter/PR-N (Sense and Anti-Sense) into pCGN1540)

The XbaI fragment of pCIB1034, which contains the chimeric PR-N gene, issubcloned into XbaI digested pCGN1540 in such a way that the 35Spromoter is proximal to the plant selectable marker. This plasmid isdesignated as pCIB1044.

The XbaI fragment of pCIB1035, which contains the chimeric PR-N gene inan anti-sense orientation, is subcloned into XbaI digested pCGN1540 insuch a way that the 35S promoter is proximal to the plant selectablemarker. The resulting plasmid is designated pCIB1045.

Example 102 Construction of pCIB1046 and pCIB1047 (Double CAMV 35SPromoter/PR-O (Sense and Anti-Sense) into pCGN1540)

The XbaI fragment of pCIB1036, which contains the chimeric PR-O gene, issubcloned into XbaI digested pCGN1540 in such a way that the 35Spromoter is proximal to the plant selectable marker. This plasmid isdesignated as pCIB1046.

The XbaI fragment of pCIB1037, which contains the chimeric PR-O gene inan anti-sense orientation, is subcloned into XbaI digested pCGN1540 insuch a way that the 35S promoter is proximal to the plant selectablemarker. The resulting plasmid is designated pCIB1047.

Example 103 Construction of pCIB1048 and pCIB1049 (Double CAMV 35SPromoter/PR-2′ (Sense and Anti-Sense) into pCGN1540)

The XbaI fragment of pCIB1038, which contains the chimeric PR-2′ gene,is subcloned into XbaI digested pCGN1540 in such a way that the 35Spromoter is proximal to the plant selectable marker. This plasmid isdesignated as pCIB1048.

The XbaI fragment of pCIB1039, which contains the chimeric PR-2′ gene inan anti-sense orientation, is subcloned into XbaI digested pCGN1540 insuch a way that the 35S promoter is proximal to the plant selectablemarker. The resulting plasmid is designated pCIB1049.

The foregoing methods can be used to insert any double 35S/cDNAexpression cassette, constructed as described in Example 73, intopCGN1540. Such a sense or antisense expression cassette includes, but isnot limited to, double 35S/PR-2″, double 35S/tobacco basicchitinase/lysozyme, and double 35S/tobacco acidic chitinase/lysozyme,double 35S/PR-4a and double 35S/PR-4b.

Example 104 Construction of pCGN1781C and pCGN1781D (Double CAMV 35SPromoter/Basic Glucanase (Sense and Anti-Sense) into pCGN1540 in EitherOrientation)

The 4.9 kb XbaI fragment of pCIB1005B is subcloned into XbaI digestedpCGN1540 in such an orientation that the double 35S promoter fragment isproximal to the plant selectable marker gene of pCGN1540 to createpCGN1787C.

The 4.5 kb XbaI fragment of pCIB1006B is subcloned into XbaI digestedpCGN1540 in such an orientation that the double 35 S promoter fragmentis proximal to the plant selectable gene of pCGN1540 to createpCGN1787D.

Example 105 Construction of pCGN1782C and pCGN1782D (Double CAMV 35SPromoter/Basic Chuitinase (Sense and Anti-Sense) into pCGN1540 in EitherOrientation)

The 4.8 kb XbaI fragment of pCGN1007 is cloned into XbaI digestedpCGN1540 in such an orientation that the double 35S promoter fragment isproximal to the plant selectable marker gene of pCGN1540 to createpCGN1782C.

The 4.8 kb fragment of pCIB1008 is cloned into XbaI digested pCGN1540,similarly, to create pCGN1782D.

Example 106 Construction of pCGN1790C and pCGN1790D (Double CAMV 35SPromoter/SAR8.2 (Sense and Anti-Sense) into pCGN1540 in EitherOrientation)

The 2.89 kb XbaI-PstI fragment of pCGN1788A is subcloned into XbaI-PstIdigested pCGN1540 to create pCGN1790C. The orientation of the double 35Spromoter in these construct is the same as the other C type constructsin the examples above.

The 2.89 kb fragment of pCGN1788B is subcloned into XbaI-PstI digestedpCGN1540 to create pCGN1790D. The orientation of the promoter in theconstruct is the same as in pCGN1790C.

Example 107 Construction of pCGN1779C, and pCGN1779D (Double CAMV35SPromoter/Cucumber Chitinase/Lysozyme (Sense and Anti-Sense) intopCGN1540 in Either Orientation)

The 4.6 kb XbaI fragment of pCIB1000 is cloned into XbaI digestedpCGN1540 in such an orientation that the double 35S promoter fragment isproximal to the plant selectable marker gene of pCGN1540 to cretepCGN1779C. The 4.6 kb XbaI fragment of pCIB1001 is cloned into XbaIdigested pCGN1540 in such an orientation that the double 35S promoterfragment is proximal to the plant selectable marker gene of pCGN1540 tocreate pCGN1779D.

Example 108 Vectors Having Hygromycin-Resistanwe as the Plant-selectableMarker Gene

Plant transformation vectors having the hygromycin-resistance geneinstead of the kanamycin gene as used above are constructed (Rothstein,et al., Gene 53: 153-161 (1987)). The vector pCIB743 is one such vector.The chimeric gene for expression in the plant is cut from any of thevectors described above using a suitable restriction enzyme, for exampleXbaI, and inserted into the polylinker of pCIB743. This constructs thechimeric gene(s) for plant expression in the broad host rangetransformation vector conferring hygromycin-resistance to transformedplant tissue. This allows one skilled in the art to utilize eitherhygromycin-resistance, or kanamycin-resistance, or both, as selectionfor transformed plant tissue.

I. STABLE TRANSFORMATION AND REGENERATION OF PLANTS

Plant tissue is transformed with the vectors described above by anytechnique known in the art. Such methods used for transfer of DNA intoplant cells include, for example, the direct infection of orco-cultivation of plants, plant tissue or cells with A. tumefaciens(Horsch, R. B. et al., Science 225: 1229 (1985); Marton, L., CellCulture and Somatic Cell Genetics of Plants 1: 514-521, 1984), treatmentof protoplasts with exogenous DNA by methods such as those described inthe following publications: Paszkowski, J. et al., EMBO J. 3: 2717(1984); EP-A 0 164 575; Shillito, R. D. et al., Bio/Technology 3: 1099(1985); Potrykus, I. et al., supra; Loerz, H. et al., Mol. Gen. Genet.199: 178 (1985); Fromm, M. et al., supra GB 2,140,822; and Negrutiu, I.et al., Plant Mol. Biol. 8: 363 (1987); incubation with polyethyleneglycol (PEG) (Negrutiu, I. et al., supra); micro-injection (Reich, T. J.et al., Bio/Technology 4: 1001-1004 (1986); Reich, T. J. et al., Can. J.Bot. 64: 1259-1267 (1986)), microprojectile bombardment (Klein, T. M. etal., Nature 327: 70 (1987)).

Example 109 Transformation of Agrobacterium With Constructs ContainingChemically Regulatable Sequences

pCIB270 Example 25), pCIB271, pCIB272 and pCIB273 (Example 28) plasmidDNA is purified on cesium chloride ethidium bromide gradients and isused to transform A. tumefaciens strain A136 containing the helperplasmid pCIB542 by the procedure of Holsters, M. et al., Mol. Gen.Genet. 163: 181-187 (1978) resulting in the strains CIB270, CIB271,CLB272 and CIB273.

Using the same procedure, plasmids pCIB271, pCIB272, and pCIB273 aretransformed into the virulent A. tumefaciens strains 15955, A208, A281,and the A. rhizogenes strain A4, creating strains designated as pCIB271X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955,pCIB272 X A208, pCIB272 X A281, pCIB272 X A4, pCB273 X 15955, pCIB273 XA208, pCIB273 X A281, and pCIB273 X A4.

Purified pCIB2001, pCIB2001/BamChit, pCIB2001/SalChit andpCIB2001/NcoChit DNA (Example 62) is used to transform Agrobacteriumtumefaciens strain CIB542 by the procedure of Holsters et al., Mol GenGenet. 163: 181-187 (1978). Agrobacterium strain CIB542 is strain EHA101(Hood et al., J. Bacteriol. 168: 1291-1301 (1986)) in which thekanamycin marker of the plasmid has been replaced by thespectinomycin/streptomycin portion of Tn7.

Example 110 Transformation of Agrobacterium tumefaciens with BinaryVectors Containing Anti-pathogenic Sequences

The binary vectors described in Section 7 are transformed intoAgrobacterium tumefaciens strain LB4404 by the following method. TheAgrobacterium strain is grown at 30° C. overnight in 5 ml of MG/L medium(50% L broth, 50% mannitol-glutamate medium (Holsters et al., Mol. Gen.Genet. 163: 4181-4187 (1978)). The 5 ml culture is added to 250 ml ofMG/L and shaken vigorously until the culture density reaches an OD=0.6at 600 nm wavelength. The cells are then collected by centrifugation at8000×g and resuspended in 5 ml of MG/L. 200 l of cells are added to 0.2to 1[B g of binary plasmid DNA in MG/L and the mix is frozen immediatelyin a dry ice/ethanol bath. After 5 minutes the tube is placed in a 37°C. water bath for 5 minutes and then 2 ml of MG/L is added. Thesuspension is kept in a 30° C. water bath for 2-3 hours and then cellare collected by centrifugation. The cells are resuspended in a minimalvolume of MG/L and then plated on selective media (MG/L plates with 100g/ml gentamycin. Colonies appear after 2-3 days at 30° C.

Example 111 General Method for Agrobacterium tumefaciens-MediatedTransformation of Nicotiana tabacum

Explains roughly 5-10 mm are cut from young leaves 3-5 cm long and thirdto sixth from the apex of Nicotiana tabacum cv ‘Xanthi nc’ grown underaxenic conditions (Facciotti and Pilet, Plant Science Letters 15: 1-7(1979) in solid MS medium (Murashige and Skoog, Physiol. Plant.15:473-497 (1962)) containing 0.7% phytagar (Gibco-BRL), 1 mg/L IAA,0.15 mg/L kinetin. These explants are plated on solid MS mediumcontaining 0.6% phytagar, 40 mg/L adenine sulfate, 2 mg/L IAA, and 2mg/L kinetin on the surface of which is placed a #1 Whatman filter andincubated for 24 hrs in the dak at 24° C. Agrobacterium strains (bearingchimeric gene constructions described above) are grown overnight in MG/Lbroth (Garfinkel and Nester, J. Bact. 144: 732-743 (1980)) at 30° C. ona shaker at 180 rmp. Explants are dipped into a bacterial suspension of3.3×10⁸ Cells/ml for approximately 5 minutes, blotted on sterile papertowels, and re-plated on the same plates.

After 48 hours explants are placed on selection medium containing thesame plate medium as above plus 350 mg/L cefotaxime and 100 mg/Lkanamycin. Co-cultivated control tissue is placed on the same medium butwithout kanamycin. The explants are transferred to fresh media every twoweeks. Shoots are harvested 4-8 weeks after co-cultivation, placed on 50ml culture tubes with 25 ml of solid MS medium containing 0.6% phytagar,1 mg/L IBA, 350 mg/L coefotaxime, and 100 mg/L kanamycin. All tissue isgrown at 24-28° C., 12 hours light, 12 hours dark light intensity 80-100Einstein. Shoots rooted in 1-2 weeks and are then transplanted toplanting mix in 4″ pots and placed in the “transgenic plant phytotron”.

Example 112 Leaf Disk Transformation of Tobacco

Agrobacterium strains containing the vectors described above are grown18-24 hours in glutamate salts media adjusted to pH 5.6 and supplementedwith 0.15% mannitol, 50 g/ml kanamycin, 50 g/ml spectinomycin and 1mg/ml streptomycin before they are diluted to an OD₆₀₀ of 0.2 in thesame media without the antibiotics. The bacteria are then grown forthree to five hours before dilution to an OD₆₀₀ of 0.2-0.4 forinoculation of discs of 5-7 mm punched from leaves of Nicotiana tabacumcv xanthi that have been grown aseptically in GA7 containers, followinga modification of the method of Horsch, R. er al., Science 227:1229-1232 (1985).

The leaf disks are maintained on 0.7% agar containing Murashige andSkoogs major and minor salts (MS), 1 mg/l benzyladenine and 1mg/ml—naphthaleneacetic acid for two days before transfer to the samemedia containing 50 g/ml kanamycin, 100 g/ml carbenicillin and 100 g/mlmefoxin. Shoots which form on the discs are excised and propagated untilsix plantlets are obtained by subculturing the shoot tips on MS mediacontaining 50 g/ml kanamycin in GA7 containers.

The plantlets are rooted on medium containing no hormones and 50 g/mlkanamycin, transferred to soil and hardened in a phytotron beforetransfer to the greenhouse for induction ment with chemical regulators.At flowering time flowers are induced to selfpollinate. Seeds ameharvested following maturation.

Example 113 Production of Transgenic Tobacco Callus and Plants

Agrobacterium strains containing the appropriate vectors are used totransform callus forming from the leaf disks (Example 112). Callusforming on kanamycin-containing MSBN selection medium is maintained on acallus growth medium comprised of MS major, minor salts and Fe-EDTA(Gibco #500-1117; 4.3 g/l), MS vitamins, 100 mg/l myo-inositol, 20 g/lsucrose, 2 mg/l naphthaleneacetic acid and 0.3 mg/l kinetin.

The callus can be used to regenerate transgenic plants by transferringcallus pieces to MSBN medium and following methods described in Examples111 and 112. The callus is also used to measure gene induction followingapplication of inducing chemicals and to screen for gene inducingchemicals.

Example 114 Transformation of Carrot

Agrobacterium strains containing the appropriate vectors as describedabove (e.g. CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 XA208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208,pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 X A208, pCIB273 XA281, and pCIB273 X A4) are grown as described in Example 112. Thebacteria, diluted to an OD₆₀₀ of 0.2-0.4, are then used for inoculationof discs cut from surface sterilized carrots.

To surface sterilize the carrots they are peeled and then soaked 20minutes in a 10% solution of chlorox. The carrots are rinsed withsterile water, sliced into 5 mm pieces and placed basal side up ontowater agar. 20-50 l of bacteria are then applied to the upper surface ofthe discs. After 7 days the discs are transferred to 0.7% agarcontaining MS salts, 3% sucrose, 0.1 mg/l 2,4-D, 50 g/ml kanamycin, 100g/ml carbenicillin, and 100 g/ml mefoxin. Callus forming around thecambial ring is excised and placed on 0.7% MS agar supplemented with 3%sucrose, 0.1 mg/l 2,4-D, 50 g/ml kanamycin, 100 g/ml carbenicillin, and100 g/ml mefoxin. After the callus has been grown it is cut into smallpieces and randomized onto four plates of the same media

For induction experiments, when the callus has filled the plate three ofthe plates are sprayed with water, 50 mM sodium salycilate pH 5.6, or250 g/l methyl benzo-1,2,3-thiadiazole-7-carboxylate to induce theexpression of the chimeric PR-1a/GUS gene.

Example 115 Transformation of Sunflower

Agrobacterium strains containing the appropriate vectors as describedabove (e.g. CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 XA208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208,pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 X A208, pCIB273 XA281, and pCIB273 X A4) are grown as described in Example 112. Thebacteria, diluted to an OD₆₀₀ of 0.2-0.4, are then used for inoculationof stems of sunflower plants prepared as follows:

Sunflower seeds are soaked 10 min in 10% captan followed by 10 min in10% chlorox and rinsing with sterile water. The seed coats are removedand the seeds are germinated on 0.7% water agar in the dark for threedays, after which they are placed into a labline incubator set at 23° C.with a 12 hour day and night. The seedlings are grown for one weekbefore decapitation and inoculation of the bacteria onto the cut stemsurface.

After one week the stems inoculated with CIB270, CIB271, CIB272 orCIB273 are cut and placed on 0.7% agar containing MS salts, 3% sucrose,2 mg/ml —napthaleneacetic acid, 1 mg/ml 6-benzylaminopurine, 100 g/mlcarbenicillin, 100 g/ml mefoxim and 50 g/ml kanamycin. Stems inoculatedwith pCIB271 X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4,pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272×A4, pCIB273 X15955, pCIB273 X A208, pCIB273 X A281, and pCIB273 X A4 are cut andplaced on 0.7% agar containing MS salts, 3% sucrose, 100 g/mlcarbenicillin, and 100 g/ml mefoxim. The callus is transferred to freshmedia every two weeks until sufficient quantity is obtained for 4plates. Half of the callus growing from the virulent Agrobacteriumstrains is transferred to media without hormones containing 50 g/mlkanamycin. After sufficient callus grown in the presence or absence ofkanamycin is obtained, it is treated for induction as described abovefor transformed carrot callus.

Example 116 Transformation of Tomato

Agrobacterium stains containing the appropriate vectors as describedabove (e.g. CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 XA208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208,pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 X A208, pCIB273 XA281, and pCIB273 X A4) are grown as described in Example 112. Thebacteria, diluted to an OD₆₀₀ of 0.2-0.4, are then used for inoculationof stems of tomato seedlings prepared as described below.

Tomato seeds are soaked 20 min in 10% chlorox and rinsed with sterilewater. The seeds are germinated on 0.7% water agar in the dark for threedays, after which they are placed into a labline incubator set at 23° C.with a 12 hour day and night The seedlings are grown for one week beforedecapitation and inoculation of the bacteria onto the cut stem surface.

After one week the inoculated stems are cut and placed on 0.7% agarcontaining MS salts, 3% sucrose, 2 mg/ml —napthaleneacetic acid, 1 mg/ml6-benzylaminopurine, 100 g/ml carbenicillin, 100 g/ml mefoxiim, and 50g/ml kanamycin. For stems inoculated with pCIB271 X 15955, pCIB271 XA208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208,pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 X A208, pCIB273 XA281, and pCIB273 X A4 the plating media used is 0.7% agar containing MSsalts, 3% sucrose, 100 g/ml carbenicillin, and 100 g/ml mefoxim. Thecallus is transferred to fresh media every two weeks until sufficientquantity is obtained for 4 plates.

For induction experimenta, half of the callus growing from the virulentAgrobacterium strains is transferred to media without hormonescontaining 50 g/ml kanamycin. After sufficient callus grown in thepresence or absence of kanamycin is obtained, it is treated forinduction as described above for transformed carrot callus.

Example 117 Transformation of Cotton

Agrobacterium strains containing the appropriate vectors (e.g. pCIB271 X15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955,pCIB272 X A208, pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 XA208, pCIB273 X A281, and pCIB273 X A4) are grown as described inExample 112. The bacteria, diluted to an OD₆₀₀ of 0.2-0.4, are then usedfor inoculation of cotton cotyledons prepared as described below.

The cotton seeds are soaked 20 min in 10% chlorox and rinsed withsterile water. The seeds are germinated on 0.7% water agar in the dark.The seedlings are grown for one week before inoculation of the bacteriaonto the cotyledon surface.

The inoculated cotyledons are allowed to form callus before they are cutand placed on 0.7% agar containing MS salts, 3% sucrose, 100 g/mlcarbenicillin, and 100 g/ml mefoxim. The callus is transferred to freshmedia every three weeks until sufficient quantity is obtained for 4plates. Half of the callus growing from the virulent Agrobacteriumstrains is transferred to media without hormones containing 50 g/mlkanamycin. For induction experiments, after sufficient callus grown inthe presence or absence of kanamycin is obtained, it is treated forinduction as described above for transformed carrot callus.

Example 118 Preparation of a Special Type of Callus of Zea Mays, EliteInbred Line Funk 2717

Zea mays plants of the inbred line Funk 2717 are grown to flowering inthe greenhouse, and self pollinated. Immature ears containing embryosapprox. 2-2.5 mm in length are removed from the plants and sterilized in10% Clorox solution for 20 minutes. Embryos are aseptically removed fromthe kernels and plated with the embryo axis downwards on OMS mediumcontainig 0.1 mg/l 2,4-D, 6% (w/v) sucrose and 25 mM L-prolinesolidified with 0.24% (w/v) Gelrite® (initiation medium). After twoweeks' culture in the dark at 27° C., the callus developing on thescutellum is removed from the embryo and plated on B5 medium, (Gamborg,O. L. et al., Experimental Cell Research 50: 151-158 (1968)), containing0.5 mg/l 2,4-D and solidified with 0.24% (w/v) Gelrite®.

The callus is subcultured every two weeks to fresh medium. Ater a totalof eight weeks after placing the embryos on the initiation medium, thespecial type of callus is identified by its characteristic morphology.This callus is subcultured further on the same medium. After a furtherperiod of two months, the callus is transferred to, and serallysubcultured on, N6 medium containing 2 mg/l 2,4-D and solidified withGelrite®.

Example 119 Preparation of a Suspension Culture of Zea Mays, EliteInbred Funk 2717

The callus described in Example 118 is subcultured for a total of atleast six months. The type of callus chosen for subculture is relativelynon-mucilaginous, granular and very friable, such that it separated intosmall individual cell aggregates upon placing into liquid medium.Cultures containing aggregates with large, expanded cells are notretained. Approximately 500 mg aliquots of the special callus of Zeamays elite inbred funk 2717 are placed into 30 ml of N6 mediumcontaining 2 mg/l 2,4-D in 125 ml Delong flasks. After one week ofculture at 26° C. in the dark on a gyratory shaker (130 rpm, 2.5 cmthrow), the medium is replaced with fresh medium. The suspensions areagain subcultured in this way after another week. At that time, thecultures are inspected, and those which did not show large numbers ofexpanded cells are retained. Suspension cultures containing aggregateswith large, expanded cells are discarded.

The preferred tissue consisted of densely cytoplasmic dividing cellaggregates which had a characteristically smoother surface than theusual type of cell aggregates. The cultures retained had at least 50% ofthe cells represented in these small aggregates. This is the desiredmorphology. These suspensions also had a rapid growth rate, with adoubling time of less than one week.

The suspension cultures are subcultured weekly by transferring 0.5 mlPCV (packed cell volume: settled cell volume in a pipette) into 25 ml offresh medium. After four to six weeks of subculture in this fashion, thecultures increased two- to three-fold per weekly subculture. Cultures inwhich more than 75% of the cells are of the desired morphology areretained for further subculture. The lines are maintained by alwayschoosing for subculture the flask whose contents exhibited the bestmorphology. Periodic filtration through 630 m pore size stainless steelsieves every two weeks is used in some cases to increase the dispersionof the cultures, but is not necessary.

Example 120 Preparation of Proloplasts from Suspension Cultures of ZeaMays

1-1.5 ml PCV of the suspension culture cells prepared as in Example 119are incubated in 10-15 ml of a filter-sterilized mixture consisting of4% (w/v) cellulase RS with 1% (w/v) Rhozyme in KMC (8.65 g/l KCl, 16.47g/l MgCl₂.6 H₂O and 12.5 g/l CaCl₂.2 H₂O, 5 g/l MES, pH 5.6) saltsolution. Digestion is carried out at 30° C. on a slow rocking table fora period of 34 hours. The preparation is monitored under an invertedmicroscope for protoplast release.

The protoplasts which are released are collected as follows. Thepreparation is filtered through a 100 m mesh sieve, followed by a 50 mmesh sieve. The protoplasts are washed through the sieves with a volumeof KMC salt solution equal to the original volume of enzyme solution. 10ml of the protoplast preparation is placed in each of several disposableplastic centrifuge tubes, and 1.5-2 ml of 0.6 M sucrose solution(buffered to pH 5.6 with 0.1% (w/v) morpholinoethane sulfonic acid (MESand KOH)) layered underneath. The tube is centrifuged at 60-100×g for 10minutes, and the protoplasts banding at the interface collected using apipette and placed in a fresh tube.

The protoplast preparation is resuspended in 10 ml of fresh KMC saltsolution, and centrifuged for five minutes at 60-100×g. The supernatantis removed and discarded, and the protoplasts resuspended gently in thedrop remaining, and then 10 ml of a {fraction (13/14)} strength KMCsolution gradually added.

After centrifuging again for five minutes, the supernatant is againremoved and the protoplasts resuspended in a {fraction (6/7)}strengthKMC solution. An aliquot is taken for counting, and the protoplastsagain sedimented by centrifugation.

The protoplasts are resuspended at 10⁷ (ten million) per ml in KM-8pmedium (Table I) or in 0.5 M mannitol containing 6 mM MgCl₂ or othersuitable medium for use in transformation as described in the followingexamples. This protoplast suspension is used for transformation and iscultured as described below in Examples 121 and 122.

Example 121 Transformation of Zea Mays Protoplasts by Electroporation

A. All steps except the heat shock are carried out at room temperature(22-28° C.). The protoplasts are resuspended in the last step of Example120 in 0.5 M mannitol containing 0.1% (w/v) MES and 6 mM MgCl₂. Theresistance of this suspension is measured in the chamber of a DialogElectroporator (DIA-LOG G.m.b.H., D-4000 Duesseldorf 13, FederalRepublic of Germany) and adjusted to 1-1.2 kOhm using a 300 mM MgCl₂solution. The protoplasts are heat-shocked by immersing the tubecontaining the sample in a water bath at 45° C. for five minutes,followed by cooling to room temperature on ice. 4 g of linearizedplasmid containing a plant-selectable hygromycin resistance gene such asdescribed by Rothstein, S. J. et al., supra or chimeric gene constructsas described in previous Examples, and 20 g of calf thymus carrier DNAare added to aliquots of 0.25 ml of this suspension. 0.125 ml of a 24%(w/v) polyethylene glycol (PEG) solution (MW 8000) in 0.5 M mannitolcontaining 30 mM MgCl₂ are added to the protoplasts. The mixture ismixed well but gently, and incubated for 10 minutes. The sample istransferred to the chamber of the electroporator and samples pulsedthree times at 10 second intervals, at initial voltages of 1500, 1800,2300 or 2800 Vcm-1, and an exponential decay time of 10 sec.

The protoplasts are cultured as follows. The samples are plated in 6 cmpetri dishes at room temperature. After a further 5-15 minutes, 3 ml ofKM-8p medium (Table I, supra) containing 1.2% (w/v) SeaPlaque agaroseand 1 mg/l 2,4-D are added. The agarose and protoplasts are mixed welland the medium allowed to gel.

B. Part A above is repeated with one or more of the followingmodifications:

(1) The resistance of the protoplast preparation is adjusted to 0.5-0.7kOhm.

(2) The PEG used is PEG with a molecular weight of 4000.

(3) No PEG is added, or one-half volume of 12% (w/v) PEG is added.

(4) The pulses are applied at intervals of three seconds.

(5) The protoplasts are plated after the electroporation in dishesplaced on a plate cooled to a temperature of 16° C.

(6) The protoplasts are placed in tubes after the electroporation step,washed with 10 ml of {fraction (6/7)} strength KMC solution or with W5solution (comprised of 380 mg/l KCl, 18.375 g/l CaCl₂.2 H₂O, 9 g/l NaCl;9 g/l glucose, pH 6.0), then collected by centrifugation at 60 g for 10minutes, resuspended in 0.3 ml of KM medium, and plated as in A.

(7) The calf thymus carrier DNA is not added.

Example 122 Transformation of Zea Mays Protoplasts by Treatment withPolyethylene Glycol (PEG)

A. The protoplasts are resuspended at the last step of Example 120 in a0.5 M mannitol solution containing 12-30 mM MgCl₂. A heat shock of 45°C. for five minutes is given as described in Example 43. The protoplastsare distributed in aliquots for transformation in centrifuge tubes, 0.3ml of suspended protoplasts per tube. During the next 10 minutes thefollowing are added: DNA (as for Example 43) and polyethylene glycol(PEG) solution (MW 6000, 40% (w/v); containing 0.1 M Ca(NO₃)₂ and 0.4 Mmannitol; pH 8-9 with KOH) to give a final concentration of 20% PEG. Thealiquots are incubated for 30 minutes with occasional gentle shaking,and then the protoplasts are placed in petri dishes (0.3 ml originalprotoplast suspension per 6 cm diameter dish) and cultured as describedin Example 121.

B. Part A above is repeated and the protoplasts are washed after 30minutes of incubation in the PEG solution of part A, by adding 0.3 ml ofW5 solution five times at two- to three-minute intervals. The protoplastsuspension is centrifuged, the supernatant removed, and the protoplastsare cultured as for Example 121, part A.

C. Parts A and B above are repeated with the modification that the finalconcentration of PEG is between 13 and 25% (w/v).

Example 123 Regeneration of Callus From Protoplasts

The plates containing the protoplasts in agarose are placed in the darkat 26° C. After 14 days, colonies arise from the protoplasts. Theagarose containing the colonies is transferred to the surface of a 9 cmdiameter petri dish containing 30 ml of N6 medium (Table I, supra)containing 2 mg/l 2,4-D, solidified with 0.24% w/v Gelrite®. This mediumis referred to as 2N6. The callus is cultured further in the dark at 26°C. and callus pieces subculturd every two weeks onto fresh solid 2N6medium.

Example 124 Selection of Transformed Callus of Zea Mays

Example 123 is repeated with the modification that 100 mg/l or 200 mg/lhygromycin B is added to the 2N6 medium in order to select fortransformed cells.

Example 125 Regeneration of Corn Plants

A. Callus is placed on 2N6 medium for maintenance and on ON6 (comprisingN6 medium lacking 2,4-D) and N61 medium (comprising N6 medium containing0.25 mg/l 2,4-D and 10 mg/l kinetin) to initiate regeneration. Callusgrowing on ON6 and N61 media is grown in the light (16 hours/day lightof 10-100 Einsteins/m²sec from white fluorescent lamps). Callus growingon N61 medium is tansfenred to ON6 medium after two weeks, as prolongedtime on N61 medium is detrimental. The callus is subcultured every twoweeks even if the callus is to be transferred again on the same mediumformulation.

Plantlets appear in about four to eight weeks. Once the plantlets are atleast 2 cm tall, they are transferred to ON6 medium in GA7 containers.Roots form in two to four weeks, and when the roots look well-formedenough to support growth, the plantlets are transferred to soil in peatpots, under a light shading for the first four to seven days. It isoften helpful to invert a clear plastic cup over the transplants for twoto three days to assist hardening off. Once the plants are established,they are treated as normal corn plants and grown to maturity in thegreenhouse. In order to obtain progeny plants are self pollinated orcrossed with wild type.

B. Part A above is repeated with the modification that 100 mg/l or 200mg/l hygromycin B is added to the medium used to maintain the callus.

Example 126 Preparation of Embryogenic Suspensions from Tissue ofDactylis Glomerata L. (Orchardgrass)

A. Embryogenic callus is initiated from basal sections of the youngestleaves of greenhouse-grown orchardgrass plants (Dactylis glomerata L.)as described by Hanning, G. E. et al., Theor. Appl. Genet., 63: 155-159(1982). The leaves are surface sterilized by immersion in a 1:10dilution of Clorox solution (5.25% (w/v) sodium hypochlorite; The CloroxCompany, Oakland, Calif.) for about 10 minutes and then cut asepticallyinto small segments of 1 to 5 mm in length or in diameter. Thesesegments are plated on sterile SH-30 medium containing 0.8% (w/v)agarose as a gelling agent. Callus and/or embryogenic structures appearwithin 2 to 6 weeks after plating, upon culture at about 25° C.Embryogenic callus is maintained by subculturing onto fresh SH-30 mediumevery 2 to 4 weeks and culturing in the dark at 25° C.

B. Embryogenic suspension cultures are initiated by placingapproximately 0.5 g fresh weight of embryogenic callus into 50 ml ofliquid medium described by Gray, D. J. et al., Plant Cell Tissue OrganCult., 4: 123-133 (1985) containing 45 M dicamba and 4 g/liter caseinhydrolysate. The suspension cultures are grown at 27° C. under a 16hours light (40 E/m ²sec), 8 hours dark photoperiod on a gyratory shakerat about 130 rpm in 125 ml Delong flasks sealed with a metal cap andparafilm®. After approximately four weeks the large clumps are allowedto settle for about 30 seconds and 10 ml aliquots of the supernatantmedium containing small cell clusters are removed and transferred to 50ml of fresh medium. This process is repeated every 3 to 4 weeks usingthe most successful cultures as judged by smaller clump size and betterquality based on the presence of small, cytoplasmic cells. After 5 to 8transfers the suspensions are essentially free of non embryogenic cellsand the majority of the embryogenic cell clusters are quite small (150to 2000 m).

Example 127 Isolation and Purification of Dactylis Glomerata L.Protoplasts

Protoplasts are prepared from embryogenic suspension cultures of Example126 by aseptically filtering the cells on a Nalgene® 0.2 m filter unitand then adding 0.5 g fresh weight cells to each 12.5 ml of protoplastenzyme mixture in a petri dish. The enzyme mixture consists of 2% (w/v)Cellulase RS, 7 mM CaCl₂×H₂O, 0.7 mM NaH₂PO₄×H₂O, 3 mM MES (pH 5.6),glucose (550 mOs/kg H₂O of pH 5.6), and is filter sterilized. Themixture is swirled on an orbital shaker at about 50 rpm in dim (<5 E/m²sec) light for about, 4 to 5 hours. The digest is then sieved through astainless steel sieve (100 m mesh size) and distributed into 12 mlcentrifuge tubes which are centrifuged at about 60 to 100 g for about 5minutes. The protoplast-containing sediment is then washed three timeswith protoplast culture medium KM-8p adjusted to 550 mOs/kg H₂O withglucose. At this point a flotation step may be included for furtherpurification of the protoplasts. In this case, the washed protoplastsare layered atop 10 ml of KM-8p culture medium adjusted 700 mOs/kg H₂Owith sucrose. After centrifugation at 60-100×g for about 10 minutes,protoplasts banding at the interface are collected using a fine pipette.Finally, the protoplasts are resuspended in 1 to 2 ml KM-8p culturemedium and sieved through a stainless mesh screen (20 m mesh size). Theprotoplasts released are collected and washed and resuspended in KM-8pmedium for culture or in osmotically adjusted medium suitable fortansformation according to the Examples below.

Example 128 Dactylis Glomerata L. Protoplast Culture and Growth ofCallus

A. The purified protoplasts are plated at a density of about 5×10⁵protoplasts per ml in KM-8p culture medium containing 1.3% (w/v)SeaPlaque® agarose (FMC Corp., Marine Colloids Division, Rockland, Me.,USA) and 30 to 40% (w/v) of conditioned medium (obtained from 3 to 4week-old Dactylis glomerata L. embryogenic suspension cultures byfiltering the medium through a sterile Nalgene® 0.2 m filter, making themedium 550 mOsm/kg H₂O by addition of glucose, and again filtersterilizing). The plates are then placed in the dark at a constanttemperature of 28° C. After 10 to 14 days the agarose is cut into wedgesand placed into ‘bead culture’ as described by Shillito, R. D. et al.,Plant Cell Reports, 2: 244-247 (1983) using 20 ml SH-45 suspensionculture medium with 3% (w/v) sucrose per 3 ml original agarose embeddedculture. The plates are put on a platform shaker and agitated at about50 rpm in light at 8 E/m ²sec. New suspension cultures are formed as thecolonies grow out of the agarose and release cells into the liquidmedium. The resultant suspension cultured cells are plated ontoagar-solidified SH-30 medium and placed in the dark at 25° C. untilcallus is formed.

B. Protoplasts are cultured as described in part A above except that theculture media contains no conditioned medium.

Example 129 Transformation of Dactylis Glomerata L. Protoplasts by Meansof Electroporation

A. Immediately after purification of the protoplasts, electroporation isperformed according to Shillito, R. D. et al., Bio/Technology 3:1099-1103 (1985) using linearized plasmid such as that as described inExample 29. The protoplasts are resuspended after the last wash at adensity of about 7×10⁶ protoplasts per ml in the electroporation buffer(0.4 M mannitol, 6 mM MgCl₂). The protoplasts are placed in 0.7 mlaliquots in 10 ml plastic centrifuge tubes.

Plasmid DNA, such as that described in Example 29, and sonicated calfthymus DNA (Sigma) to give final concentrations of 10 g/ml and 50 g/mlrespectively is added to the tubes. Then 0.38 ml polyethylene glycol(PEG) solution [24% (w/v) PEG 6000 in 0.4 M mannitol 30 mM MgCl₂, 0.1%(w/v) MES (pH 5.6)] is added and the solution gently mixed. Theprotoplast suspension is transferred into the chamber of a Dialog®Electroporator and 10 pulses of 3250 V/cm initial voltage andexponential decay constant of 10 sec applied at 30 sec intervals. Thesample is removed from the chamber, and placed in a 10 cm diameter petridish. 10 ml of KM-8p medium containing 1.2% (w/v) SeaPlaque® agarose isadded, the protoplasts distributed evenly throughout the medium, and theagarose allowed to gel.

B. Part A above is repeated except that the initial voltage used is 3500V/cm, 4000 V/cm, 5000 V/cm, 3000 V/cm, or 2500 V/cm.

C. Parts A and B above are repeated except that PEG of MW 4000 or PEG ofMW 8000 is used.

D. Parts A to C are repeated except that the final PEG concentration isbetween 10% and 30% (w/v).

Example 130 Transformation of Dactylis Glomerata L. Protoplasts byTreatment with Polyethylene Glycol (PEG)

A. PEG medicated direct gene transfer is performed according toNegrutiu, I. et al., supra. The DNA used is linerized plasmid such asthat described in Example 29.

The protoplasts are suspended following the last wash in 0.5 M mannitolcontaining 15 mM MgCl₂ at a density of about 2×10⁶ per ml. Theprotoplast suspension is distributed as 1 ml aliquots into 10 ml plasticcentrifuge tubes. The DNA is added as described in Example 51 above, andthen 0.5 ml of the PEG solution added (40% (w/v) PEG 4000 in 0.4 Mmannitol, 0.1 M Ca(NO₃)₂, pH 7.0). The solutions are mixed gently andincubated for 30 minutes at room temperature (about 24° C.) for 30minutes with occasional shaking. 1.4 ml of the wash solution is thenadded, and the contents of the tube gently mixed. The wash solutionconsists of 87 mM mannitol, 115 mM CaCl₂, 27 mM MgCl₂, 39 mM KCl, 7 mMTris-HCl and 1.7 g/l myo-inositol, pH 9.0. Four further 1.4 ml aliquotsof wash solution are added at 4 minute intervals, with mixing after eachaddition. The tube is then centrifuged at about 60×g for about 10minutes, and the supernatant discarded. The sedimented protoplasts aretaken up in 1 ml KM-8p culture medium, and placed in a 10 cm petri dish.10 ml of KM-8p medium containing 1.2% (w/v) SeaPlaque® agarose is added.The protoplasts are evenly distributed throughout the medium, and theagarose allowed to gel.

B. Part A is repeated with one or more of the following modifications:

(1) The pH of the wash solution is adjusted to 5.6 or 7.0.

(2) The PEG used is PEG of MW 6000, PEG of MW 2000 or PEG of MW 8000.

(3) The wash medium consists of 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 5mM glucose, pH to 6.0 with KOH, of 0.2 M CaCl₂, 0.1% (w/v) MES, pH 6.0with KOH, or of 0.2 M CaCl₂, 7 mM Tris/HCl, pH 9.0 with KOH.

Example 131 Transformaton of Dactylis Glomerata L. Protoplasts byEletroporation or PEG Treatment

Transformation is carried out as described in Examples 129 or 130,except that the protoplasts are treated at 45° C. for about 5 minutesprior to distribution of the aliquots into tubes for transformation orafter distribution of the aliquots, and before addition of the PEG.

Example 132 Selection of Transformed Colonies

A. The culture plates (petri dishes) containing the protoplasts fromExamples 129 to 131 are incubated for 10 days in the dark at about 25°C. and then cut into 5 equal slices for ‘bead cultures’ (Shillito, R. D.et al., Plant Cell Reports 2: 244-247 (1983)). Four of the slices areplaced each into 20 ml SH-45 culture medium with 4 g/l caseinhydrolysate and 20 g/ml hygromycin B. The fifth slice is put into 20 mlof the same medium but without hygromycin B as a non-selected control.After 4 to 5 weeks the putative transformed protoplast-derived cellcolonies growing in hygromycin B are cut out of the agarose and placedinto a 19 mm petri dish with 2 ml of liquid SH-45 medium containing 20g/ml hygromycin B, which is agitated at about 50 rpm on an orbitalshaker. After another 4 to 5 weeks all colonies which grow to make newsuspensions are transferred into 125 ml Erlenmeyer flasks and grown in amanner similar to the parent suspension culture, except that 20 g/mlhygromycin B is included in the medium.

The new suspensions are subcultured every 1 to 3 weeks using SH-45medium containing 4 g/l casein hydrolysate and 20 g/ml hygromycin B.Cells from these suspensions are also plated on solidified SH-30 mediumcontaining 20 g/ml hygromycin B and incubated at about 25° C. in thedark. Calli grown from the plated cells are subcultured every two weeksonto fresh medium. The cells which grow in the presence of hygromycin Bare presumed to be transformants.

B. Selection is carried out as described in part A except that theprotoplast-derived cell colonies growing in hygromycin B containingmedium are placed on agar plates of SH-30 medium containing 20 g/mlhygromycin B and incubated at about 25° C. in the dark.

Example 133 Regeneration of Transformed Dactylis Glomerata L. Plants

A. Dactylis Glomerata L. callus (obtained as described in Example 132)derived from protoplasts is grown on solidified SH-30 medium, andsubcultured every two weeks. Any embryos which form are removed andplated on germination medium (SH-0) and placed in the light (45 to 55E/m ²sec). Germination of these embryos occurs in 1 to 4 weeks and theresultant plantlets are placed on SH-0 medium in the light to form rootsystems. They are moved into the greenhouse at the six to twelve leafstage, and hardened off gradually.

B. Callus (obtained as described in Example 132) derived fromprotoplasts is grown on SH-0 medium solidified with 0.24% (w/v) Gelrite®in the light (45 to 55 E/m ²sec), and subcultured every two weeks. Theresultant plantlets are placed on a 1:1 mixture of SH-0 and OMS mediasolidified with a combination of 0.12% (w/v) Gelrite® and 0.4% (w/v)agar in the light to form root systems. They are moved to the greenhouseat the six to twelve leaf stage, and hardened off gradually.

C. Small plantlets are obtained as described in Example 125, parts A andB, and are placed on OMS medium solidified with 0.8% (w/v) agar in thelight to form root systems. They are moved to the greenhouse at the sixto twelve leaf stage, and hardened off gradually.

D. Small plantlets are obtained as described in Example 125, part A andare placed on a 1:1 mixture of SH-0 and OMS media solidified with acombination of 0.12% (w/v) GelRite® and 0.4% (w/v) agar in the light toform root systems. They are moved to the greenhouse at the six to twelveleaf stage, and hardened off gradually.

J. TRANSIENT GENE EXPRESSION

The following examples describe how the chimeric genes can be introducedand utilized without the gene necessarily being stably incorporated intothe genome of the plant.

Example 134 Introduction of DNA into Protoplast of N. Tabacum byTreatment with PEG

A. Preparation of protoplasts of N. tabacum can be carried out inaccordance with the methods described in the following publications:Paszkowski, J. et al., EMBO J. 3: 2717 (1984); GB patent application 2159 173, European Patent Application EP 0 129 668; Shillito, R. D. andPotrykus, I., Methods in Enzymology 153: 313-306, (1987) or by othermethods known in the art.

B. DNA is introduced into protoplasts by a modification of the method ofNegrutiu, I. et al., Plant Mol. Biol. 8: 363 (1987). The protoplastsprepared as described in part A are resuspended following the lastwashing step in a solution consisting of 0.4 M mannitol, 15-30 mM CaCl₂,0.1% w/v MES at a density of 1.6-2×10⁶ per ml. The protoplast suspensionis distributed as 0.5 ml aliquots into 10 ml plastic centrifuge tubes.DNA such as that described in Examples 28 to 40 is added in 10 l steriledistilled water, sterilized as described by Paszkowski, J. et al., EMBOJ. 3: 2717 (1984), and then 0.5 ml of the PEG solution (40% (w/v) PEG MW8000 in 0.4 M mannitol, 0.1 M Ca(NO₃)₂, pH 7.0) is added. The solutionsare mixed gently and incubated for 30 minutes at room temperature (about24° C.) with occasional shaking. 1 ml of the wash solution is thenadded, and the contents of the tube gently mixed. The wash solutionconsists of 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, pH to 6.0with KOH. Further aliquots of 2 ml, 3 ml and 4 ml of wash solution areadded sequentially at 5 minute intervals, with mixing after eachaddition. The tube is then centrifugated at about 10-100×g for about 10minutes, and the supernatant discarded. The pelleted protoplasts aretaken up in sufficient K3 culture medium with 0.3 M glucose as theosmoticum, and no sucrose, to achieve a final density of 100,000 per mland cultured in a 10 cm petri dish.

C. Part B is repeated with one or more of the following modifications:

(1) The pH of the wash solution is adjusted to 5.6 or 7.0.

(2) The PEG used is PEG with a molecular weight of 4000.

(3) The wash medium consists of 0.2 M CaCl₂, 0.1% w/v MES, pH 6.0 withKOH, or of 0.2 M CaCl₂, 7 mM Tris/HCl, pH 9.0 with KOH.

(4) 50 g of sheared calf thymus DNA in 25 l sterile water is addedtogether with the plasmid DNA.

(5) The plasmid DNA is linearized before use by treatment with anappropriate restriction enzyme (e.g. BamHI).

Example 135 Introduction of DNA into Protoplasts of N. Tabacum byElectroporaion

A. Introduction of DNA into protoplasts of N. tabacum is effected bytreatment of the protoplasts with an electric pulse in the presence ofthe appropriate DNA, in a modification of the methods of Fromm, M. E.,Methods in Enzymology 153: 307, (1987); and Shillito R. D and PotrykusI., ibid., p. 283-306.

Protoplasts are isolated as described in Example 134, part A. Theprotoplasts are resuspended following the last wash in the followingsolution: 0.2 M mannitol, 0.1% w/v MES/72 mM NaCl, 70 mM CaCl₂, 2.5 mMKCl, 2.5 mM glucose, pH to 5.8 with KOH, at a density of 1.6-2×10⁶ perml. The protoplast suspension is distributed as 1 ml aliquots intoplastic disposable cuvettes and 10 g of DNA added as described inExample 134, parts B and C. The resistance of the solution at this pointwhen measured between the electrodes of the 471 electrode set of theelectroporation apparatus described below is in the range of 6 Ohms.

DNA such as that described in Examples 28 to 40 is added in 10 l steriledistilled water, sterilized as described by Paszkowski, J. et al., EMBOJ. 3: 2717 (1984).The solution is mixed gently and then subjected atroom temperature (24-28° C.) to a pulse of 400 V/cm with an exponentialdecay constant of 10 ms from a BIX-Transfector 300 electroporationapparatus using the 471 electrode assembly. The protoplasts are leftundisturbed for 5 minutes, and then placed in a petri dish and K3 mediumas described in Example 56 A added to bring the density of protplasts to100,000 per ml.

B. Part A is repeated with one or more of the following modifications:

(1) The voltage used is 200 V/cm, or between 100 V/cm and 800 V/cm.

(2) The exponential decay constant is 5 ms, 15 ms or 20 ms.

(3) 50 g of sheared calf thymus DNA in 25 l sterile water is addedtogether with the plasmid DNA.

(4) The plasmid DNA is linearized before use by treatment with anappropriate restriction enzyme (e.g. BamHI).

Example 136 Introduction of DNA into Protoplasts of Zea Mays Line 2717

Protoplasts of maize inbred line Funk 2717 are prepared as described inExamples 118 to 125 above, and resuspended in in either of the solutionsdescribed for resuspension of the N. tabacum protoplasts in Examples 134and 135 above at a density of 10⁷ per ml. Transformation is carried outessentially as described in Examples 134 and 135. The protoplasts arecultured following transformation at a density of 2×10⁶ per ml in KM-8pmedium with no solidifying agent added and containing 1 mg/l 2,4-D.

Example 137 Introduction of DNA into Protoplasts of Sorghum Bicolor

Protoplasts of sorghum suspension FS 562 are prepared essentially asdescribed for Zea mays in Example 118 above, and resuspended in ineither of the solutions described for resuspension of the N. tabacumprotoplasts in Examples 134 and 135 above at a density of 10⁷ per ml.Transformation is carried out essentially as described in Example 136.The protoplasts are cultured following transformation at a density of2×10⁶ per ml in KM-8p medium, with no solidifying agent added.

Example 138 Introduction of DNA into Protoplasts of NicotianaPlumbaginifolia, Petunia Hybrida and Lolium Multiflorum

Protoplasts of N. plumbaginifolia, P. hybrida or L. multiflorum areprepared as described in Shillito and Potrykus, supra, and treated asdescribed in Examples 134 and 135. They are cultured in the mediumdescribed by Shillito and Potrykus, supra, without addition of agaroseor any other gelling agent.

Example 139 Introduction of DNA into Protoplasts of Glycine Max

Protoplasts of Glycine max are prepared by the methods as described byTricoli, D. M. et al., Plant Cell Reports 5: 334-337 (1986), orChowhury, V. K. and Widholm, J. M., Plant Cell Reports 4: 289-292(1985), or Klein, A. S. et al., Planta 152: 105-114 (1981). DNA isintroduced into these protoplasts essentially as described in Examples134 and 135. The protoplasts are cultured as described in Klein, A. S.et al., supra, Chowhury V. K. and Widholm, J. M. supra, or Tricoli, D.M. et al., supra, without the of addition of alginate to solidify themedium.

K. CHEMICAL REGULATION OF TRANSGENIC PLANT TISSUE

The following examples describe procedures for the chemical regulationof chemically regulatable genes in representative transgenic planttissue. While the following examples are directed to induction, similarprocedures would be applicable for systems operating by repression.

A variety of techniques are available for the treatment of transgeniccells with a chemical regulator. For example, cells may be suspended inliquid medium containing the regulator. Callus may be grown on, ortransferred to medium containing the regulator, or the regulator may beapplied to callus with a sterile paint brush or plant mister. Likewiseplants or parts of plants are treated with a solution or a suspension ofthe regulator by the way of a paint brush or, preferably, a plantmister.

Expression of the phenotypic trait in transgenic plants containing achemically regulatable chimeric gene is proportional to the amount ofthe regulator applied to the plant or plant tissue.

Example 140 Chemical Induction in Transgenic Callus Cultures

A. Callus obtained according to previously described procedures isassayed for chemical induction by applying with a sterile paint brush ora plant mister a filter sterilized solution of 0.1 to 50 mM salicylicacid which has been adjusted to a pH between 6.0 and 8.0 by the additionof NaOH.

After induction the callus is allowed to incubate for two days and thenharvested, frozen in liquid nitrogen and stored at −80° C. until theyare assayed for gene induction as described in Example 65 and/or Example66.

B. Alternatively, the following solutions are used for induction:

(1) 0.1 to 50 mM benzoic acid which has been adjusted to a pH between6.0 and 8.0 by the addition of NaOH.

(2) 0.1 to 50 mM polyacrylic acid which has been adjusted to a pHbetween 6.0 and 8.0 by the addition of NaOH.

(3) Methyl benzo-1,2,3-thiadiazole-7-carboxylate. The solution is madeby resuspending a wettable powder containing the chemical in sterilewater for several minutes at a concentration of 1 mg/nm. Insoluble solidis removed from the solution by filter sterilizing.

(4) 0.1 to 50 mM benzo-1,2,3-thiadiazole-7-caboxylic acid, pH between6.0 and 8.0. The compound is dissolved in a minimum of dimethylsulfoxide (DMSO), then diluted with sterile water to the desiredconcentration. The obtained suspension is applied without filtering.

(5) 0.1 to 50 mM n-propyl benzo-1,2,3-thiadiazole-7-carboxylate,prepared as in (4).

(6) 0.1 to 50 mM benzyl benzo-1,2,3-thiadiazole-7-carboxylate, preparedas in (4).

(7) 0.1 to 50 mM benzo-1,2,3-thiadiazole-7-carboxylic acidN-sec-butylhydrazide, prepared as in (4).

(8) 0.1 to 50 mM 2,6-dichloroisonicotinic acid, pH between 6.0 and 8.0,prepared as in (4).

(9) 0.1 to 50 mM methyl 2,4-dichloroisonicotinate, prepared as in (4).

Example 141 Chemical Induction in Transgenic Plants

A. Gene expression is induced in plants obtained according to previouslydescribed procedures by applying a fine spray of a solution of 0.1 to 50mM salicylic acid which has been adjusted to a pH between 6.0 and 8.0with NaOH, to the leaves using a plant mister.

The plants are allowed to continue to grow for two days and are thenharvested, frozen in liquid nitrogen and stored at −80° C. until theyare assayed as described in Example 65 and/or Example 66.

B. Alternatively, the following solutions are used for induction:

(1) 0.1 to 50 mM benzoic acid which has been adjusted to a pH between6.0 and 8.0 by the addition of NaOH.

(2) 0.1 to 50 mM polyacrylic acid which has been adjusted to a pHbetween 6.0 and 8.0 by the addition of NaOH.

(3) Methyl benzo-1,2,3-thiadiazole-7-carboxylate. The solution is madeby resuspending a wettable powder containing the chemical in sterilewater for several minutes at a concentration of 1 mg/ml. Insoluble solidis removed from the solution by filter sterilizing.

(4) 0.1 to 50 mM benzo-1,2,3-thiadiazole-7-carboxylic acid, pH between6.0 and 8.0. The compound is dissolved in a minimum of dimethylsulfoxide (DMSO), then diluted with sterile water to the desiredconcentration. The obtained suspension is applied without filtering.

(5) 0.1 to 50 mM n-propyl benzo-1,2,3-thidiazole-7-carboxylate, preparedas in (4).

(6) 0.1 to 50 mM benzyl benzo-1,2,3-thiadiazole-7-carboxylate, preparedas in (4).

(7) 0.1 to 50 mM benzo-1,2,3-thiadiazole-7-carboxylic acidN-sec-butylhydrazide, prepared as in (4).

(8) 0.1 to 50 mM 2,6-dichloroisonicotinic acid, pH between 6.0 and 8.0,prepared as in (4).

(9) 0.1 to 50 mM methyl 2,6-dichloroisonicotinate, prepared as in (4).

Example 142 Induction of Expression of the Introduced DNA in Protoplastsof N. Tabacam, Zea Mays, N. Plumbagnifolia P. Hybrida, L. Multiflorumand Sorghum Bicolor

The protoplasts treated as described in Examples 134 to 139 above arecultured at 26° C. in the dark for 2 days. After this time salicylicacid is added to a concentration of between 0.1 to 50 mM as aneutralized solution, pH between 6.0 and 8.0. The protoplasts arecultured for a further 2 days, harvested by centrifgation, quick frozenand assayed as described in the following Examples.

B. Alternatively, neutralized solutions or suspensions of the followingcompounds are added to the protoplasts to give a final concentration ofbetween 0.1 and 50 mM:

(1) benzoic acid

(2) polyacrylic acid

(3) methyl benzo-1,2,3-thiadizole-7-carboxylate

(4) benzo-1,2,3-thiadiazole-7-carboxylic acid

(5) n-propyl benzo-1,2,3-thiadiazole-7-carboxylate

(6) benzyl benzo-1,2,3-thiadiazole-7-carboxylate

(7) benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide

(8) 2,6-dichloroisonicotinic acid

(9) methyl 2,6-dichloroisonicotinate

C. Alternatively, induction is carried out as described in parts A and Bexcept that the protoplasts are cultured for 1 day, 3 days or 5 daysbefore induction.

D. Induction is carried out as described in Examples parts A, B or Cexcept that the protoplasts are cultured for 1 day, 3 days or 5 daysafter induction and before being harvested.

Example 143 Assay for Chemically Inducible DNA Sequence: mRNA Production

Plant materials induced and harvested as in Examples 140 to 142 areassayed for the induction of mRNA species by the isolation of RNA andthe primer extension assay as described in Example 6.

Callus or regenerated plant material derived from transgenic plantscontaining chemically regulatable chimeric genes coding for GUS, AHAS orBT and induced by salicylic acid, methylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid, n-propylbenzo-1,2,3-thiadiazole-7-caxboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide,2,6-dichloroisonicotinic acid, methyl 2,6-dichloroisonicotinate, or TMVdemonstrates elevated levels of mRNA upon assay with the GUS, AHAS or BTprimer, respectively, in the primer extension assay. The level of mRNAis proportional to the amount of regulator applied.

Example 144 Assay for Chemically Inducible DNA Sequences:β-Glucuronidase Enzymatic Activity A. ELISA Assay

Frozen leaf tissue is ground in a mortar with a pestle in the presenceof liquid nitrogen to produce a fine powder. Leaf extracts are preparedby mixing a given weight (g) with an equal volume (ml) of GUS extractionbuffer (50 mM sodium phosphate buffer pH 7.0, 0.1% Triton-X 100, 0.1%sarkosyl, 10 mM β-mercaptoethanol) as described by Jefferson, R. A. etal., Proc. Natl. Acad. Sci. USA 83: 8447-8451 (1986).

The reactions are carried out in the wells of ELISA plates by mixing5-25 l extract with 120-100 l GUS assay buffer (50 mM sodium phosphatebuffer pH 7.0, 0.1% Triton X-100, 10 mM β-mercaptoethanol) containing4-methyl umbelliferyl glucuronide (MU) at a final concentration of 2 mMin a total volume of 125 l. The plate is incubated at 37° C. for 1-5hours and the reaction is stopped by the addition of 150 l 3 M Na₂CO₃.The concentration of fluorescent indicator released is determined byreading the plate on a Flow Labs Fluoroskan II ELISA plate reader.

B. Results

TABLE II Specific Activity nKat MU/mg protein Plant^(a) A B C D E^(b) H1 5.39 ± 13.9 ± 13.5 ± 20.1 ± 33.6 ± 1.9  1.2  0.8  5.1  2.6  H 3 0.21 ±6.92 ± 11.95 ± 1.66 ± 6.54 ± 0.02 0.88 0.85  0.02 143 H 4 8.39 ± 27.5 ±18.52 ± 2.28 ± 25.1 ± 1.31 0.7  1.07  0.78 0.9  H 6 0.10 ± 4.08 ± 2.58 ±0.09 ± 2.8 ± 0.04 0.58 .01  0.06 1.2 H 9 0.23 10.8 9.1 0.5 7.5 Alu 1 5.620.0 206.9 20.5 70.5 Alu 2 2.0 6.6 83.2 8.9 155.3 Alu 3 0.1 ± .07 2.8 ±.9  2.0 ± .9  2.0 ± .5  5.8 ± 3.8 Alu 4 2.1 ± 1   43.0 ± 7.1 ± 2.6 6.1 ±3.2 7.5 ± 3.5 19  Alu 5 0.6 ± .2  4.4 ± 2.3 1.2 ± .6  0.5 ± .3  8.3 ±3.7 Alu 6 0.8 28.8 67.0 3.1 36.4 Alu 7 1.7 38.7 29.1 21.7 63.0 Alu 8 0.60.4 0.4 0.6 0.2 Alu 9 0.9 0.6 0.4 0.3 nd Alu 10 4.0 39.2 52.0 8.1 37.9Alu 11 1.6 ± .8  1.9 ± .9  2.8 ± 1.3 2.2 ± 2.6 2.8 ± 2.9 Alu 12 0.7 ±.4  57.1 ± 73.4 2.3 ± 1.2 78.0 ± 22   41.4 Alu 13 0.1 19.1 13.2 1.9 10.6Hae 1 0.6 1.6 2.6 1.7 3.9 Hae 2 2.3 8.8 11.8 0.4 0.8 Hae 3 1.7 11.1 11.23.0 1.6 Hae 4 2.0 8.1 10.8 1.7 0.82 Hae 5 18.1 59.4 132.5 36.6 14.0 Hae8 0.4 30.6 28.9 0.6 29.0 Hae 9 2.6 63.9 129.7 12.5 55.2 Hae 10 1.6 70.598.2 13.5 94.5 Hae 11 2.6 81.8 58.5 3.9 46.0 Hae 12 0.2 2.5 5.8 0.6 14.5Hae 13 0.01 0.7 0.1 nd nd Hae 14 0.1 1.8 8.6 0.1 3.3 Hae 15 0.1 6.4 7.23.4 7.4 Hae 16 0.1 2.3 4.3 0.8 2.7 ^(a)H 1-9 are tobacco plants obtainedby transformation of leaf disks by strain CIB271 Alu 1 -13 are tobaccoplants obtained by transformation of leaf disks by strain CIB272 Hae1-16 are tobacco plants obtained by transformation of leaf disks bystrain CIB273 ^(b)A Water B Salicylic acid C Methylbenzo-1,2,3-thiadiazole-7-carboxylate D Buffer E TMV

Transformed tobacco callus (Example 113): The following GUS activitiesare obtained from samples of transformed callus which has been harvested21 hours after treatment. Transformed with CIB271: H₂O spray: 1.8 nKatMU/mg protein; 50 mM sodium salicylate spray: 3.8 nKat MU/mg protein.

Transformed with CIB271 (different experiment): H₂O spray: 2.8 nKatMU/mg protein; 250 g/l methyl benzo-1,2,3-thiadizole-7-carboxylatespray: 13.7 nKat MU/mg protein. Transformed with CIB273: H₂O spray: 0.46nkat MU/mg protein; 250 g/l methyl benzo-1,2,3-thiadiazole-7-carboxylatespray: 31.2 nKat MU/mg protein.

Transformed carrot (Example 114): The following GUS activities areobtained from samples of callus transformed by pCIB271 which has beenharvested 21 hours after treatment. H₂O spray: 5.2 pKat MU/mg protein.50 mM sodium salicylate spray: 11.6 pKat MU/mg protein. 250 g/l methylbenzo-1,2,3-thiadiazole-7-carboxylate spray: 22.1 pKat MU/mg protein.

Transformed tomato (Example 116): The following GUS activities areobtained from samples of transformed callus which has been harvested 21hours after treatment. Transformed with CIB271: H₂O spray: 17.2 pKatMU/mg protein; 250 g/l methyl benzo-1,2,3-thiadiazole-7-carboxylatespray: 56.1 pKat MU/mg protein. Transformed with CIB273 X A4: H₂O spray:4.6 pKat MU/mg protein; 250 g/l methylbenzo-1,2,3-thiadiazole-7-carboxylate spray: 22.1 pKat MU/mg protein.

Transient expression in tobacco protoplasts (Example 134) treated withpCIB269: H₂O: 30.6 13 pKat MU/mg protein; methylbenzo-1,2,3-thiadiazole-7-carboxylate: 66.6 20 pKat MU/mg protein.

Example 145 Assay for Chemical Induction of the Endogenous Gene

As a control procedure, tobacco plant materials induced as described inExamples 139-141 by salicylic acid, methylbenzo-1,2,3-thiadiazole-7-carboxylate and TMV are assayed fortranscription of mRNA from the endogenous PR-1a gene by the methodsdescribed in Example 6. Elevated levels of mRNA are detected afterinduction by all three inducers.

Example 146 Assay for Chemically Inducible DNA Sequences:Acetohydroxyacid Synthase (AHAS) Enzymatic Activity A. SamplePreparation for AHAS Assay

The weight of the leaf and/or root tissue material to be assayed isdetermined, the tissue is placed into liquid nitogen and macerated to afine powder. One volume (in ml) of cold homogenization buffer (50 mMNaH₂PO₄ buffer pH 7.0, 0.5 mM EDTA pH 7.0, 0.5 mM MgCl₂, 1 mM sodiumpyruvate pH 7.0, 10 M flavine adenine dinucleotide, 1 mMphenyl-methylsulfonyl fluoride, 10% glycerol) equal to two times theweight (in g) of tissue is added and the mixture is transferred to aFrench pressure cell. All subsequent steps are carried out keeping thematerial cold (about 4° C.). The cells are disrupted at 16,000 psi,collecting the cell exudate into a container on ice. Alternatively thetissue is ground with homogenization buffer in a mortar and pestle inplace of the French pressure cell treatment. The exudate is centrifuged5 min at 10,000 rpm to clear the cell debris. The volume of the supemateis measured and enzyme grade ammonium sulfate added to 25% saturation(144 mg (NH₄)₂SO₄/ml supernate). The mixture is stirred on ice for 15min, then centrifuged for 5 min at 10,000 rpm. The pellet is discardedand the supernate adjusted to 50% saturation with ammonium sulfate (158mg (NH₄)₂SO₄/ml supemnate). After stirring on ice for 15 min, the pelletis collected by centrifugation at 10,000 rpm for 5 min. The pelletobtained is dissolved in 1 ml of column buffer for each 2 g of leaf or 5g of root material. The extract is desalted by application to a SephadexG50 column equilibrated with column buffer (50 mM NaH₂PO₄ buffer pH 7.0,0.1 mM EDTA, 0.5 mM MgCl₂, 10 M flavine adenine dinucleotide, 1 mMphenylmethylsullfonyl fluoride, 10% glycerol). The sample is eluted withcolumn buffer, collecting 2 ml of sample for each ml of sample appliedto the column.

B. Tube Assay

5-50 l of an extract prepared according to part A are dissolved in atotal volume of 0.5 ml of reaction mixture (20 mM NaH₂PO₄ buffer pH 7.0,20 mM sodium pyruvate pH 7.0, 0.5 mM thiamine pyrophosphate, 5 mM MgCl₂,10 M flavine adenine dinucleotide). The mixture is incubated for 30 minat 37° C. The reaction is stopped by the addition of 50 l of 6N H₂SO₄.The mixture is incubated for 15 min at 60° C., then treated with 625 lof—napthol reagent (500 l 5%—napthol in 2.5 M NaOH and 125 l 25 mg/mlcreatine) and incubated another 15 min at 60° C. If a precipitate hasformed, this is spun out, and the absorbance measured at 520 m. ThepMoles acetoin formed are calculated from a standard curve developedusing 0.4 g to 10 g acetoin. The specific activity is expressed aspKat/mg protein.

C. Elisa Plate Assay

1-20 l of a sample are placed in an Elisa plate well with a total volumeof 0.15 ml of reaction mixture. The mixture is reacted for 30 min at 37°C. The reaction is stopped by the addition of 50 l of 2.4 M H₂SO₄contaning 1% creatine. The mixture is incubated for 30 min at 60° C. Anyprecipitate present is separated by centrifugation. 150 l of the assaymixture are transferred to a new Elisa plate, treated with 100 l10%—napthol in 5M NaOH and incubated another 20 min at 60° C. Theabsorbance is measured at 520 m, and the specific activity expressed asunder part B.

Example 147 Assay for Chemicaly Inducible DNA Sequences: Bacillusthuringiensis Endotoxin A. Plant Extraction Procedure

About 100 mg plant tissue is homogenized in 0.1 ml extraction buffer (50mM Na₂CO₃ pH 9.5, 10 mM EDTA, 0.05% Triton X-100, 0.05% Tween, 100 mMNaCl, 1 mM phenylmethylsulfonyl fluoride (added just prior to use), and1 mM leupeptine (added just prior to use)). After extraction, 2 M TrispH 7.0 is added to adjust the pH to 8.0-8.5. The extract is thencentrifuged 10 minutes in a Beckman microfuge and the supernatant usedfor ELISA analysis.

B. ELISA

An ELISA plate is pretreated with ethanol. Affinity-purified rabbitanti-Bt antiserum (50 l) at a concentration of 3 g/ml in borate-bufferedsaline (100 mM boric acid, 25 mM sodium borate, 75 mM NaCl, pH 8.4-8.5)is added to the plate and this allowed to incubate overnight at 4° C.Antiserum is produced by immunizing rabbits with gradient-purified Bt(Bacillus thuringiensis endotoxin) crystals (Ang, B. J. & Nickerson, K.W., Appl. Environ. Microbiol. 36: 625-626 (1978)) solubilized withsodium dodecyl sulfate. The plate is washed with wash buffer (10 mMTris-HCl pH 8.0, 0.05% Tween 20, 0.02% sodium azide), then treated 1hour at room temperature with blocking buffer (10 mM sodium phosphatebuffer pH 7.4, 140 mM NaCl, 1% bovine serum albumin, 0.02% sodiumazide), and washed again. The plant extract is added in an amount togive 50 g of protein (typically ca. 5 l of extract; the protein isdetermined by the method of Bradford, M., Anal. Biochem. 72: 248 (1976)using a commercially available kit from Bio-Rad), and incubatedovernight at 4° C. After washing, 50 l affinity-purified goat anti-Btantiserum are added at a concentration of 3 g/ml protein in ELISAdiluent (10 mM sodium phosphate buffer pH 7.4, 140 m NaCl, 0.05% Tween20, 1% bovine serum albumin, 0.02% sodium azide), and this allowed toincubate for 1 hour at 37° C. After washing, 50 l rabbit anti-goatantibody bound to alkaline phosphatase (Sigma Chemicals, St. Louis, Mo.,USA) diluted 1:500 in ELISA diluent is added and allowed to incubate for1 hour at 37° C. After washing, 50 l substrate (0.6 mg/ml p-nitrophenylphosphate, 0.05 mg/ml MgCl₂, 10% diethanolamine, pH 9.8 adjusted withHCl) is added and allowed to incubate for 30 minutes at roomtemperature. The reaction is terminated by adding 50 l 3 M NaOH. Theabsorbance is read at 405 nm in a modified ELISA reader (HewlettPackard, Stanford, Calif., USA).

Example 147A Analysis of Cucumber Chitinase Constructs A. TransgenicPlant Treatments

Once the transgenic plants are transferred to soil, they are grown 4-5weeks before treatment. The following solutions are applied to theleaves with sterile paintbrushes:

1. Sterile dH2O

2. 50 mM salicylic acid

3. Methyl benzo-1,2,3-thiadiazole-7-carboxylate. The solution is made byresuspending a wettable powder containing a formulation of the chemicalwith 25% active ingredient in sterile water at a concentration of 1mg/ml.

4. Methyl 2,6-dichloroisonicotinate, prepared as above. Alternatively,30 ul of 10 mM sodium phosphate buffer, pH 7.0, or 30 ul of the samebuffer containing tobacco mosaic virus (50-100 pfu/30 ul) is applied tothe leaves which have been sprinkled with carborundum. The buffers aredistributed over the whole treated leaf by gentle rubbing.

The plants are allowed to continue to grow for seven days. The treatedleaves are then harvested, frozen in liquid nitrogen and stored at −80°C. until they are assayed.

B. ELISA Assay

Frozen leaf tissue is ground in a mortar with a pestle in the presenceof liquid nitrogen to produce a fine powder. 0.33 g of each tissuesample is added to 1 ml extraction buffer (50 mM Tris-HCl, pH 8.5, 0.2M2-mercaptoethanol, 2 mM phenyl methyl sulfonyl fluoride, 2 mMbenzamidine, 10 mM epsilon-aminocaproic acid, 1 mM leueptine) and thesample homogenized for one minute with an Ultra Turrax Mixer (shaft#S25N-8G, Tekmar Co. Cincinnati, Ohio). The samples are then centrifugedat 48,000×g for fifteen minutes at 4° C. and the supernatant removedfrom the pelleted material. The extracts are diluted at least 1:50 inELISA diluent (10 mM sodium phosphate, pH 7.4, 140 mM sodium chloride,0.05% Tween 20, 0.02% sodium azide, 1% BSA).

A microtiter plate is coated with monoclonal antibody specific to thecucumber chitinase protein at 2 ug/ml in borate-buffered saline (100 mMboric acid, 25 mM sodium borate, 75 mM sodium chloride; pH 8.5) using 50ul per well. The plate is incubated overnight at 4° C. The plate iswashed with ELISA wash buffer (10 mM Tris-HCl, pH 8.0, 0.05% Tween 20, 3mM sodium azide) and BSA blocking solution (10 mM sodium phosphate, pH7.4, 140 mM sodium chloride, 0.02% sodium azide, 1% BSA) is added tocompletely cover each well. Incubation is for at least 30 minutes atroom temperature.

The microtiter plate is washed with ELISA wash buffer and 50 ul ofextract is added to the appropriate well. 50 ul of each of 10 chitinaseprotein standards (ranging from 0 ng/ml to 31.64 ng/ml) was also addedto the appropriate well and the plate is incubated for two hours at 4°C. The plate is washed with ELISA wash buffer and 50 ul of rabbitpolyclonal antibody to the cucumber chitainase protein (diluted 1:15,000in ELISA diluent) is added to the well and incubated for one hour at 37°C. After washing with ELISA wash buffer, 50 ul of goat anti-rabbitconjugated to alkaline phosphatase (1:500 in ELISA diluent) is added toeach well and incubated at 37° C. for one hour. The plate is then washedand 50 ul alkaline phosphatase substrate solution (0.6 mg p-nitrophenylphosphate in 0.5 mM magnesium chloride and 10% diethanolamine) is addedto each well. After a 30 minute incubation at room temperature, 50 ul of3N sodium hydroxide is added to each well to stop the reaction.

The plate is read at 405 nm and 492 nm on an HP Genenchem ELISA platereader. The output values are expressed as A(405 nm)—A(492 nm)—0.092 toallow for correction of p-nitrophenyl phosphate as substrate. Thechitinase standards for each plate are graphed logarithmically. Theamount of chitinase protein in each sample is determined from thestandard curve. If the amount of chitinase in any sample is close to theupper limit of the curve, the sample is assayed again at a greaterdilution. Final results are expressed in the table below in ug chitinaseper gram tissue.

TABLE I TRANSGENIC CHITINASE PLANTS - ELISA ASSAY RESULTS (ugchitinase/g tissue) TREATMENTS Salicy- MB- MD- LINE Water late TH7CBuffer TMV CINA 2001/BamChit Bam-1 0.066 — 3.000 — 33.885 — Bam-3 — —0.504 — 4.227 — Bam-4 — — — — — — Bam-8 — — — — 2.244 — Bam-9 — 0.52233.885 — 28.255 — Bam-10 0.059 0.093 25.182 0.084 102.436 2.378 Bam-12 —— 1.349 — 2.421 — Bam-14 — — 0.541 — 7.818 — Bam-15 — — 3.366 — 7.964 —Bam-16 — 0.690 18.849 — 17.803 0.864 Bam-17 — — 2.997 — 6.041 — Bam-19 —0.760 0.998 — 28.255 — Bam-21 — — — — 2.260 — Bam-23 — — 0.782 — 2.421 —Bam-24 — — 0.88 — 12.081 — Bam-25 — 0.522 18.145 — 3.389 — Bam-26 — —0.676 — 4.676 — 2001/SalChit Sal-2 — — 0.117 — — Sal-3 — — 0.060 — 0.1060.047 Sal-4 — 0.047 0.537 — 4.740 — Sal-5 — — 0.229 — 0.288 — Sal-7 — —0.377 — 0.600 — Sal-8 — — — — 0.117 — Sal-9 — 1.052 0.553 — 5.478 —Sal-11 — — — — — — Sal-12 — — — — 0.189 — Sal-13 — — — — 0.047 — Sal-14— — — — — — Sal-15 — — 0.038 — 1.999 — 2001/NcoChit Nco-2 — — — — — —Nco-3 — — — — — — Nco-5 — — — — — — Nco-8 — — — — — — Nco-9 — — — — — —Nco-10 — — 0.047 — — — Nco-11 — — — — — — Nco-15 — — — — — — Nco-160.555 0.555 0.784 0.566 0.784 0.286 Nco-17 — — — — — — —Indicates thatsample has no detectable level of chitinase protein

C. Transformation of the Chimeric Construct

The plasmid pCIB2001/Chit/GUS is transfonmed into Agrobacterium aspreviously described in Example 109. Transformed Agrobacterium strainsare prepared and transformed into tobacco leaf disks as previouslydescribed in Example 112. Transformed plants treatments and harvestingare as described in section A above.

D. Beta-Glucuronidase Enzyme Assay

Frozen leaf tissue is ground in a mortar with a pestle in the presenceof liquid nitrogen to produce a fine powder. Leaf extracts are preparedin GUS extraction buffer (50 mM sodium phosphate pH7.0, 0.1% Triton-X100, 0.1% sarkosyl, 10 mM beta-mercaptoethanol) as described byJefferson, R. A. et al., PNAS USA 83, 8447-8451 (1986).

The reactions are carried out in the wells of microtiter plates bymixing 5-25 ul of extract with 120-100 ul of GUS assay buffer (50 mMsodium phosphate pH 7.0, 0.1% Triton X-100, 10 mM beta-mercaptoethanol)containing 4-methyl umbelliferyl glucuronide (MU) at a finalconcentration of 2 mM in a total volume of 125 ul. The plate isincubated at 37° C. for 1-5 hours and the reaction is stopped by theaddition of 150 ul 3 M sodium carbonate. The concentration offluorescent indicator released is determined by reading the plate on aFlow Labs Fluoroskan II ELSA plate reader.

The amount of protein in each extract is determined using the BCAProtein Assay (Pierce, Rockford, Ill.) according to the manufacturer'srecommendations. The specific activity is determined for each sample andcan be expressed in nKat MU/mg protein.

Example 147B Chemical Induction of the Arabidopsis PR-1 Promoter

Leaves of transgenic Arabidopsis lines carrying the PR-1 promoter-LUCgene fusion were treated by spraying with 0.15 g/ml INA (isonicotinicacid) for 48 h. Five days later, sprayed leaves were analyzed forluciferase activity using the Promega Luciferase Assay System (Cat. #E1500). Transgenic lines showed 144-fold induction over controls. Inother assays, luciferase activity was measured by imaging lines forbioluminescence using intensified cameras (VIM and photon-counting imageprocessors (ARGUS-50 or ARGUS-100) from Hamamatsu Photonic Systems(Bridgewater, N.J.). Leaves were sprayed with INA and subsequently witha 5 mM solution of D-luciferin (Analytical Bioluminescence Laboratories,San Diego, Calif.) 24 h before and then immediately before imaging.Transgenic plants carrying the PR-1-LUC fusion had strongly inducedbioluminescence when compared to water-treated controls.

L. ANALYSIS OF TRANSGENIC PLANTS CONTAINING ANTI-PATHOGENIC SEQUENCES

In the previous sections, the creation of transgenic plants expressingchimeric disease resistance genes has been described. In this sectionthe development of transgenic seed lines and characterization of thoselines with respect to chimeric gene expression is explained.Essentially, this characterization process comprises a preliminaryscreening of the transgenic plants for expression of the chimeric gene,segregation of the chimeric gene into stable homozygous lines andfurther characterization of the gene expression.

Example 148 Development of Transgenic T3 Seed Lines

Genotype designations for transgenic plants are used herein according tothe following convention: the initial plant resulting from atransformation event and having grown from tissue culture is designateda T1 plant. Plants resulting from self pollination of the naturalflowers of the T1 plant, arn designated T2, having acquired a newgenotype during the normal meotic process. likewise, seeds borne fromself-pollination of the natural flowers of T2 plants (i.e. grown from T2seed) are designated T3, etc. Transgenic plants (T1) are grown tomaturity. Flowers are allowed to self-pollinate and seed pods arecollected after normal desiccation. Seeds from each individual plant arecollected and stored separately. Each seed lot is tested by geneticsegregation analysis to determine the number of Mendelian loci bearingthe kanamycin resistance trait. T2 seeds are surface-sterilized bymultiple washing in 2% hypochlorite containing 0.02% (v/v) Tween-20,followed by rinses in sterile water. Approximately 150 of the seeds areplaced on filter paper saturated with 0.2×MS salts (Murashige and Skoog,Physio. Plant. 15: 473-497 (1962)) containing 150 g/ml kanamycin.Following germination and expansion of the cotyledons to approximately,5 mm, the ratio of normal-green (kan-r) versus bleached (kan-s)cotyledons is determined. Only those T2 seed lots exhibiting anapproximately 3:1 (kan-r:kan-s) ratio are kept for further analysis;this segregation ratio is indicative of a single Mendelian locus bearingthe kanamycin marker gene.

Four to ten plants are grown to maturity from each T2 seed lot (usingthe same conditions described above), and are allowed to self-pollinate.T3 seed collection, seed sterilization, and seed germination are asdescribed above for the T2 seed. T3 seed lots in which 100% of thetested seeds (n=150) exhibited the kan-r phenotype are assumed to behomozygous for the trait (i.e. resulting from a homozygous T2 parentplant) and ame kept for phenotypic analysis.

Example 149 Expressing Sense or Anti-sense PR-1

The expression of PR-1a in either sense or anti-sense orientation isassayed in transgenic plant mateial using either an ELISA assay forPR-1a protein or a primer extension assay for PR-1 mRNA as described.

A. ELISA for PR1 Protein

Assays are performed in Immunolon II microtiter plates (Dynatech) whichhad been rinsed with ethanol and allowed to air dry. Tobacco leafmaterial is ground with a plastic tissue homogenizer (Kontes) in abuffer consisting of 50 mM Tris-HCl, pH 8.5, 200 mM 2-mercaptoethanol, 2mM PMSF (Sigma), 2 mM BAM (Sigma), 10 mM ACA (Sigma), and 0.048% (w/v)Leupeptine (hemisulfate salt) (Sigma); three ml of extraction buffer areused per gram of leaf tissue. A sufficient sample of healthy-leaf(untreated tobacco) extract is made so that a {fraction (1/10)} dilutionof this extract could serve as a diluent for all the other samples.Extracts are centrifuged in a microcentrifuge in 1.5 ml polypropylenetubes at 12,000×g (max) for 15 minutes to remove debris. Wells arecoated with a solution of a monoclonal antibody specific for PR1 protein(tobacco). Following washing the wells are blocked for 30-120 minuteswith a solution of bovine serum albumin (1% w/v) and then washed again.

The unknown samples and the standard curve samples, diluted toappropriate concentrations in a {fraction (1/10)} solution ofhealthy-plant extract, are added to the wells and incubated for 1 hourat 37° C. (A standard curve is performed using highly purified PR-1aprotein). Following washing, a rabbit polyclonal antiserum (5 g/mlspecific for PR1 is added to the wells and incubated for an additionalhour at 37° C., and then the wells are washed again. A goat, anti-rabbitIgG antibody (133 ng/ml), to which is conjugated the indicator enzymealkaline phosphatase (Promega), is added and the indicator reaction isdeveloped according to the manufacturer's recommendations. The reactionis stopped after 30 minutes by the addition of NaOH and the absorbenceis read at 405 nm.

B. Primer Extension Assay for PR1

RNA is extracted from tobacco leaf tissue by a method previouslydescribed (Ecler and Davis, Proc. Natl. Acad. Sci, USA 84: 5203-5206(1987)). Primer extension assays are performed as described in Example 6using a synthetic oligonucleotide of the sequence5′GTAGGTGCATTGGTTGAC3′. The complement of this sequence occurs in bothPR-1a and PR-1b mRNA, resulting in priming of both types of mRNA in theassay.

The primer extension products of the chimeric gene product aredistinguishable from the products of the endogenous PR-1 genes usingpolyacrylamide gel electrophoresis. The chimeric PR-1a transcriptgenerated from the tobacco RuBISCO small subunit promoter of pCGN1509derivatives results in a primer extension product which is 90 bp longerthan that of the endogenous PR-1a gene. The chimeric PR-1b transcriptgenerated from the tobacco RuBISCO small subunit promoter of pCGN1509derivatives produces a primer extension product 95 bp longer than thatof the endogenous PR-1b gene. The chimeric PR-1a transcript generatedfrom the double 35S promoter of pCGN1761 derivatives yields a product 4bp longer than that of the endogenous PR-1a gene. The chimeric PR-1btranscript generated from the double 35S promoter of pCGN1761derivatives yields a product 10 bp longer than that of the endogenousPR1b gene.

Example 150 Analysis of Seed Lines Derived From Transformaton of TobaccoWith pCGN1755 and pCGN1756 Series Vectors (RuBISCO SSU/PR-1a orPR-1b/ocs 3′)

A leaf tissue sample is taken from T1 plants transformed with either:pCGN1754 or pCGN1760 (as empty cassette controls); one of the pCGN1755binary vector series (SSU promoter/PR-1a in all orientations); or one ofthe pCGN1756 binary vector series (SSU promoter/PR-1b). The expressionof PR-1 protein in this tissue is determined by ELISA for PR-1 proteinand in some cases the RNA level is monitored by primer extension assay.

It is predicted that tissue transformed with the control plasmids,pCGN1754 and pCGN1760 (empty cassette) will result in a certain basallevel of PR-1 protein which would be due to endogenous synthesis. Tissuetransformed with the plasmids pCGN1755A, pCGN1755C, pCGN1756A orpCGN1756C (PR-1a or PR-1b in a sense orientation) should produce plantsexpressing PR-1 at a level significantly higher than the basal level.Tissue transformed with pCGN1755B, pCGN1755D, pCGN1756B or pCGN1756D(PR-1a or PR-1b in an anti-sense orientation) should producesignificantly lower levels of PR-1 protein relative to the control. Whenthe transformed T1 tissue is screened for PR-1 protein by ELISA thoseplants which conform to the expectation are promoted to T2 analysis. Theintent in this screening is to eliminate transformants that do notcontransform the chimeric PR-1 gene with the antibiotic resistance gene.Many plants from each transformation do conform to the expectation andthe protein result is confirmed by primer extension analysis of the RNAto make sure that the higher level of expression in sense plants is dueto chimeric gene expression.

The assays are repeated at the T2 generation and at this point severallines are chosen for further characterization. The lines are chosen forbased on: 1) a 3:1 segregation of the antibiotic resistance, whichindicates a Mendelian inherited (single insertion event) trait; 2) highlevels of expression of PR-1 for sense construct, low levels foranti-sense constructs and intermediate levels for control plants. TheLines chosen for further study and the data for PR-1 expression andsegregation are shown below.

T2 Segregation T2 PR-1 expression Seed Line Analysis (% Kan-R) ELISA(ng/ml) 1754-12 77% 300 1755A-4 75% 175 (Av = 515 in T3) 1755B-2 83% ≦21755B-3 76% ≦2 1760-1 93% 240

The seed line nomenclature is designed such that the transformingplasmid is designated first and the individual transformant isdesignated second. For example, 1755A-4 represents a transgenic plantresulting from transformation with the plasmid pCGN1755A and this is thefourth individual transformant selected. In many of these experimentsthe control level of PR-1 expression seems artificially high (ie 1754-12above at a level of 300 ng/ml is about 30 times higher than normal).This level decreases in the control, but not in the experimental, plantswith successive generations.

The T2 seed lines above are mixed genotypes in respect to the chimericPR-1 gene. For instance, some of the plants from a seed line arehomozygous and some are heterozygous for the trait. In order to isolatehomozygous seed lines, between four and ten plants of each line areallowed to self pollinate and set seed. This seed is collected andsegregation is determined as explained above. These segregation data forseveral lines is shown below.

T3 Seed Line Segregation (% Kan-R) 1754-12-10 100 1755A-4-2 1001755B-2-1 100 1755B-3-1 100 176O-1 ND

These homozygous seed lines are then analyzed for generalized diseaseresistance as described below. The general conclusion from the analysisof this series of plants is that the sense constructs (both PR-1a andPR-1b) produce 6 to 150 times the amount of PR-1 in healthy tobaccotissue and anti-sense constructs usually produce much less PR-1 than inhealthy tobacco.

Example 151 Analysis of Seed Lines Derived From Transformation ofTobacco With pCGN1764, pCGN1765, pCGN1774 and pCGN1775 Seres of Vectors(Double CAMV 355 Promoter/PR-1a)

The development of seed lines in this example include thetransformations of tobacco with the double CAMV 35S promoter linked toPR-1a (pCGN1764 and pCGN1774 series) in sense and anti-sense orientationand the double CAMV 35S promoter linked to PR-1b (pCGN1765 and pCGN1775series) in sense and anti-sense orientation. The difference between thepCGN1764/pCGN1765 and pCGN1774/pCGN1775 constructs is that the binaryvector is different (see relevant examples above). Empty cassettecontrols for pCGN1764 and pCGN1765 are pCGN1766 and pCGN1767. The emptycassette control for pCGN1774 and pCGN1775 is pCGN1789.

PR1 protein expression in the “senses” T1 plants (all events) range fromundetectable levels up to approximately 13,000 ng/ml extract; thismaximum level is within two fold of the levels seen in a highly infectedprimary leaf bearing many lesions. The levels seen in secondary tissue,even under optimal conditions, is several fold lower than this. Theaverage expression level for all the “sense” T1 plants is approximately,4,200 ng/ml, which is more than 20-fold higher than the average for thesmall subunit-PR1 transgenic plants.

No significant differences are seen in the expression levels of thechimeric gene between the pCGN783 binary vector (pCGN1764 and pCGN1765series). Both sets of plants have a wide range of expression levels, asis common in transgenic experiments of this type. The highest expressionin both types is similar, and the number of plants expressing at a lowlevel is about the same. The average for the pCGN783 binary plants ishigher than the average for the small subunit-PR1 transgenic plants.

No significant differences are seen in the expression levels of thechimeric gene between the pCGN783 binary vector (pCGN1764 and pCGN17065series) and the pCGN1540 binary vector (pCGN1774 and pCGN1775 series).The average for the pCGN783 binary plants is 3,955 ng/ml, and for thepCGN1540 binary plants is 4,415 ng/ml, but considering the variation,this difference is not significant. Similarly, the orientation of thegenes in the binary has no major effect on expression. The “C”orientation (head to tail) gives three of the four highest expressingplants, but it also gives more low level expressing plants. The “A”(head to head) orientation plants tend to group more in the moderateexpression range, but again the variation and the small sample sizeprevent the attachment of any statistical significance to thesedifferences. Primer extension analysis of a limited number of samplesshows that the chimeric gene mRNA is the dominant or the only PR1 mRNApresent.

The conclusion from the T1 data is that the level of PR-1 protein inplants transformed with double CAMV 35S promoter/PR-1a or PR-1b senseconstructs is several hundred fold higher than the level of controlplants. The level of expression of PR-1 in plants transformed with theanti-sense construct is very low. The binary vector used fortransformation (pCGN783 or pCGN1540) does not significandy effect thelevel of PR-1 expression in the transgenic plants. Likewise, theorientation of the expression cassette within the vector has nosignificant effect on the level of PR-1 expression. Therefore, one lineis selected that produces high levels of PR-1a due to sense expressionand one is selected that produces low levels of PR-1a due to anti-senseexpression for further development. A control line with an emptycassette is included. The results of PR-1 expression and antibioticsegregation for the selected lines is shown below.

Segregation T2 PR-1 expression T2 Seed Line Analysis (% Kan-R) ELISA(ng/ml) 1774A-10 409/553 (74.0) 9000 1774B-3 109/142 (76.8) ≦2 1789-10371/459 (80.8) 5.6

Homozygous T3 seed lines are generated from each of the selected linesas described in the previous example. The results of segregationanalysis are shown below.

Segregation T3 Seed Line (% Kan-R) 1774A-10-1 100 1774B-3-2 1001789-10-3 100

These homozygous seed lines are evaluated for PR-1 expression anddisease resistance as described below.

Example 152 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1779 Plasmid Series (Double CAMV 35SPromoter/Cucumber Chitinase/Lysozyme)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1779C or pCGN1779D. The cucumberchitinase/lysozyme protein content is determined using an ELISA assayessentially as described above except that the monoclonal and polyclonalantibodies are directed against the cucumber chitinase/lysozyme protein.

Eight of thirteen T1 “sense” plants produce very high amounts (>10,000ng/ml extract) of the cucumber chitinase foreign gene product. Again awide range, from undetectable up to 31,500 ng/ml extract, is observed,with an average of 12,500 ng/ml extract. The conclusion from the T1 datais that the transformed T1 plants produce several thousand times more ofthe transgenic protein than is present in control plants. T3 seed linesare derived from the high expressing T1 plants as described in Example148 and these T3 seed lines maintain their high levels ofchitinase/lysozyme expression.

Example 153 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1782 Plasmid Senes (Double CAMV 35SPromoter/Tobacco Basic Chitinase)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1782C or pCGN1782D. The tobacco basic chitinaseprotein content is estimated by an immunoblot technique (Towbin, H, etal., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979) as modified byJohnson, D., et al., Gene Anal. Tech. 1: 3-8 (1984)), followingSDS-polyacrylamide gel electrophoresis (Laemmli, E., Nature, 227:680-685 (1970)). The antibodies used are raised against the tobaccobasic chitinase protein by standard methods and are specific for thetobacco basic chitinase protein. T1 plants with the pCGN1782C plasmid(containing the sense expression cassette) showing high levels ofexpression relative to control and anti-sense plants, are advanced to T3seed lines as described in Example 148. Homozygous T2 plants which yieldthese T3 seed continue to express the protein at high levels. T1 plantstransformed by the pCGN1782D plasmid (containing the anti-senseexpression cassette) which give low levels of expression are alsoadvanced to T3 seed as described in Example 148.

Example 154 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1781 Plasmid Series (Double CAMV 35SPromoter/Tobacco Basic Glucanase)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1781C or pCGN1781D. The tobacco basic glucanaseprotein content is estimated by an immunoblot technique as described inExample 153 above. The antibodies used are raised against the tobaccobasic glucanase protein by standard methods and are specific for thetobacco basic glucanase protein. T1 plants with the pCGN1781C plasmid(containing the sense expression cassette), showing high levels ofexpression relative to control and anti-sense plants, are advanced to T3seed lines as described in Example 148. Homozygous T2 plants which yieldthese T3 seed continue to express the protein at high levels. T1 plantstransformed by the pCGN1781D plasmid (containing the anti-senseexpression cassette) which give low levels of expression are alsoadvanced to T3 seed as described in Example 148.

Example 155 Analysis of Seed Lines Derived From Transformaton of TobaccoWith the pCGN1783 Plasmid Series (Double CAMV 35S Promoter/Tobacco PR-Rmojor)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1783C or pCGN1783D. The tobacco PR-R proteincontent is estimated by an immunoblot technique as described in Example153 above. The antibodies used are raised against the tobacco PR-Rprotein by standard methods and are specific for the tobacco PR-Rprotein. T1 plants with the pCGN1783C plasmid (containing the senseexpression cassette) showing high levels of expression relative tocontrol and anti-sense plants, are advanced to T3 seed lines asdescribed in Example 148. Homozygous T2 plants which yield these T3 seedcontinue to express the protein at high levels. T1 plants transformed bythe pCGN1783D plasmid (containing the anti-sense expression cassette)which give low levels of expression are also advanced to T3 seed asdescribed in Example 148.

Example 156 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1790 Plasmid Series (Double CAMV 35S Promoter/SAR8.2)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1790C or pCGN1790D. The SAR8.2 protein content asestimated by an immunoblot technique as described in Example 153 above.The antibodies used are raised against the SAR 8.2 protein by standardmethods and are specific for the SAR 8.2 protein. T1 plants with thepCGN1790C plasmid (containing the sense expression cassette) showinghigh levels of expression relative to control and anti-sense plants, areadvanced to T3 seed lines as described in Example 148. Homozygous T2plants which yield these T3 seed continue to express the protein at highlevels. T1 plants transformed by the pCGN1790D plasmid (containing theanti-sense expression cassette) which give low levels of expression arealso advanced to T3 seed described in Example 148.

Example 157 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1791 Plasmid Series (Double CAMV 35S Promoter/PR-Q)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1791C or pCGN1791D. The PR-Q protein content isestimated by an immunoblot technique as described in Example 153 above.The antibodies used are raised against the PR-Q protein by standardmethods and are specific for the PR-Q protein. T1 plants with thepCGN1791C plasmid (containing the sense expression cassette) showinghigh levels of expression relative to control and anti-sense plants, areadvanced to T3 seed lines as described in Example 148. Homozygous T2plants which yield these T3 seed continue to express the protein at highlevels. T1 plants transformed by the pCGN1791D plasmid (containing theanti-sense expression cassette) which give low levels of expression arealso advanced to T3 seed as described in Example 148.

Example 158 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1792 Plasmid Series (Double CAMV 35SPromoter/PR-Q′)

A leaf tissue sample is taken from T1 plants trasformed with either ofthe binary vectors pCGN1792C or pCGN1792D. The PR-O′ protein content isestimiated by an immunoblot technique as described in Example 153 above.The antibodies used are raised against the PR-O′ protein by standardmiethods and are specific for the PR-O′ protein. T1 plants with thepCGN1792C plasmnid (containing the sense expression cassette) showinghigh levels of expression relative to control and anti-sense plants, areadvanced to T3 seed lines as described in Example 148. Homozygous T2plants which yield these T3 seed continue to express the protein at highlevels. T1 plants transformed by the pCGN1792D plasmid (containing theanti-sense expression cassette) which give low levels of expression arealso advanced to T3 seed as described in Example 148.

Example 159 Analysis of Seed Lines Derived From Transformation ofTobacco With the pCGN1793 Plasmid Senes (Double CAMV 35S Promoter/PR-2)

A leaf tissue sample is taken from T1 plants transformed with either ofthe binary vectors pCGN1793C or pCGN1793D. The PR-2 protein content isestimated by an immunoblot technique as described in Example 153 above.The antibodies used are raised against the PR-2 protein by standardmethods and are specific for the PR-2 protein. T1 plants with thepCGN1793C plasmid (containing the sense expression cassette) showinghigh levels of expression relative to control and anti-sense plants, areadvanced to T3 seed lines as described in Example 148. Homozygous T2plants which yield these T3 seed continue to express the protein at highlevels. T1 plants transformed by the pCGN1793D plasmid (containing theanti-sense expression cassette) which give low levels of expression arealso advanced to T3 seed as described in Example 148.

11. EVALUATION OF PHENOTYPE

The development of stable, transgenic seed lines of tobacco whichexpress chimeric pathogenesis-related protein genes in sense andanti-sense orientation is explained in Section 1 to 8. Once the seedlines are developed they are evaluated quantitatively for resistance tovarious diseases.

Example 160 Evaluation of Transgenic Tobacco Expressing PR-1 in anAnti-Sense Orientation for Disease Resistance

The seed lines 1755A-4-2 and 1755B-2-1 are analyzed for resistance toTMV. The results of these experiments are that there is no significantdifference in lesion size or lesion number due to either elevated ordepressed levels of PR-1 protein.

The seed lines 1755A-4-2 and 1755B-2-1 are analyzed for resistance tothe fungal pathogen Peronospora tabacina (blue mold, or downy mildew) byspraying a spore suspension on the leaves of the plants and incubatingunder standard conditions for seven days. The plants are then scored forresistance to bluemold based on the percentage of leaf surface areainfected by the pathogen. Six plants of the 1755A-4-2 line which areexpressing an average of 1454 ng/ml PR-1 protein are showing 98±3%infected surface area. Six plants of the 1755B-2-1 line, which areexpressing an average of 370 ng/ml PR-1 protein are showing 45%±26%infected surface area. Six plants derived from untransformed Xanthi.nctobacco which are producing 559 ng/ml PR-1 protein are showing 99%±1%infected surface area. This result indicates that the anti-senseexpression of PR-1a results in a significant and valuable resistance todowny mildew in transgenic plants.

Example 161 Evaluation of Transgenic Tobacco Expressing PR-1 in a SenseOrientation for Disease Resistance

The seed lines 17745A-10-1 and 1774B-3-2 are analyzed for resistance toTMV. The results of these experiments are that there is no significantdifference in lesion size or lesion number due to either elevated ordepressed levels of PR-1 protein.

The seed lines 1774A-10-1 and 1774B-3-2 are analyzed for resistance tothe fungal pathogen Peronospora tabacina (blue mold, or downy mildew) byspraying a spore suspension on the leaves of the plants and incubatingunder standard conditions for seven days. The plants are then scored forresistance to bluemold based on the percentage of leaf surface areainfected by the pathogen. Six plants of the 1774A-10-1 line are showing6.3%±11% infected surface area. Six plants of the 1774B-3-2 line areshowing 46%±10% infected surface area. Six plants derived fromuntransformed Xanthi.nc tobacco are showing 55%±5% infected surfacearea. This result indicates that the sense expression of PR-1a resultsin a significant and valuable resistance to downy mildew in transgenicplants.

Example 162 Generation of F1 Plants Containing Two of the Above ChimenrcGenes by Genetic Crossing

Using standard techniques, T3 homozygous plants expressing differentcDNAs are cross-pollinated in all possible combinations. The totalnumber of pariwise gene combination is calculated from the expression

(n²⁻n)/2

where n=number of parental T3 lines. For instance, for the case of sixseparate T3 parental lines, each expressing a different cDNA, the numberof crosses required to yield all possible combinations of two expressedcDNAs is 15. In addition, all crosses are performed reciprocally, i.e.each T3 parental line is used as both male (pollen) parent and female(ovule) parent for each cross.

Example 163 Evaluation of Transgenic Tobacco Expressing Basic Chitinasein a Sense and Anti-Sense Orientation for Disease Resistance

The seed lines 1782C (sense orientation) and 1782D (anti-senseorientation) are analyzed for resistance to the bacterial pathogenPseudomonas tabaci. Suspensions of bacteria (10⁶⁻3×10⁶/ml) are injectedinto the intercellular space of the leaves. The plants are incubatedunder 100% relative humidity at 20° C. for three days and then firtherincubated at 40% relative humidity. The symptoms caused by the bacteriaare evaluated 3,4,5 and 6 days after the inoculation as follows:

0=no symptom

1=1% to 25% of the injected area shows symptoms (yellowing or necrosis)

2=26% to 50% of the injected area shows symptoms (yellowing or necrosis)

3=51% to 75% of the injected area shows symptoms (yellowing or necrosis)

4=76% to 99% of the injected area shows symptoms (yellowing or necrosis)

5=100% of the injected area shows symptoms (yellowing or necrosis) 1test, 3×10⁶b/ml

disease severity Plant 3 day 4 day 5 day 6 day non-transformed 3.5 ± 1.23.6 ± 1.2 4.0 ± 1.3 4.5 ± 0.9 empty cassette 1.5 ± 1.4 2.3 ± 1.5 2.4 ±1.4 2.5 ± 1.4 chitinase sense 1.0 ± 0.8 1.6 ± 1.1 1.4 ± 0.5 1.4 ± 1.0chitinase 3.9 ± 0.8 4.4 ± 0.7 4.5 ± 0.9 4.8 ± 0.4 antisense

As there is a high difference between the symptom severity on thechitinase sense and antisense transformed plants as well as between thenon-transformed plants and empty cassette, the test is repeated.

disease severity Plant 3 day 4 day 5 day 2nd test, 3 × 10⁶ b/mlnon-transformed 3.8 ± 0.9 4.0 ± 1.0 4.1 ± 1.0 empty cassette 3.8 ± 0.74.2 ± 0.7 4.2 ± 0.8 chitinase sense 2.3 ± 1.0 3.2 ± 1.1 2.4 ± 1.3chitinase 4.8 ± 0.4 5.0 ± 0.0 5.0 ± 0.0 antisense 2nd test, 10⁶b/mlnon-transformed 1.7 ± 0.8 1.5 ± 0.8 1.5 ± 0.8 empty cassette 2.4 ± 1.02.2 ± 1.0 1.8 ± 1.1 chitinase sense 1.4 ± 0.5 1.4 ± 0.5 0.9 ± 0.2chitinase 3.8 ± 0.4 3.8 ± 0.4 3.4 ± 1.0 antisense

In this second test, the enhanced resistance of the chitinase-senseplants and the enhanced susceptibility of the chitinase antisense plantsis clear.

Example 164 Evaluation of Transgenic Tobacco Expressing CucumberChitinase or Tobacco Basic Chitinase for Disease Resistance

The seed line 1779C (sense orientation) from Example 152 (tobaccotransformed with pCGN1779 plasmid (Double CAMV 35S promoter/cucumberchitinase) is analyzed for resistance to damping off, a disease causedby the soil fungal pathogen Rhizoctonia solani.

The following plants are grown:

a) control plants grown in uninfected soil

b) transgenic plants expressing the PR-Q

c) control plants transformed with an empty cassette

d) transgenic plants expressing the tobacco basic chitinase gene.

e) transgenic plants expressing cucumber chitinase.

The plants transformed with tobacco basic chitinase cucumber chitinaseexhibit enhanced resistance to damping off.

Example 165 Evaluation of Transgenic Tobacco Expressing SAR8.2 in aSense Orientation for Disease Resistance

The seed lines 1790C (sense orientation) (Example 156) is analyzed forresistance to black shank, a disease caused by the fungal pathogenPhytopthora.

Four control plants are transformed with an empty expression cassetteand are severely affected by black shank disease.

Four plants are transformed with the chimeric double 35S-SAR8.2construct and exhibit appreciable enhanced resistance to black shankdisease.

Example 166

Analysis of Seed Lines Derived From Transformation of Tobacco with theBasic and Acidic Class III Tobacco Chitinase Genes

Binary constructions carrying the tobacco basic and acidic class IIIchitinases were transformed into Nicotiana tabacum cv Xanthi-nc. Leaftissue samples were taken from T1 plants and assayed for expression ofeither transgene protein (by Western analysis) or transgene RNA (byNorthern analysis). T1 plants found to express the transgene at highlevels were advanced to T3 seed lines as described above.

A. Analysis of Transgenic Plants Expressing Class III Chitinase for PestResistance

Transgenic plant lines expressing one or more class III chitinase genesare assessed for resistance to numerous pests. Approximately six plantsof each line are tested. Pests at the appropriate stage of their growthcycle (such as larvae) are introduced at the appropriate stage of plantdevelopment. Plants are later assayed for % of leaf or tissue areaeaten, % of introduced larvae surviving, and the weight of survivinglarvae.

B. Analysis of Transgenic Tobacco Plants Expressing Class III Chitinasefor Pest Resistance

Transgenic tobacco plants expressing class III chitinase are assessedfor resistance to numerous pests. Those tested include Spodoptera exigua(beet armyworm), the green peach tobacco aphid, Manduca sexta, andvarious nematodes, weevils, mites, and other pests.

C. Analysis of Transgenic Plants Expressing Basic Class III Chitinasefor Resistance to Helothis virescens

Transgenic tobacco lines expressing the tobacco basic class IIIchitinase gene were assessed for resistance to the insect Heliothisvirescens. Eight plants of each line were tested. 50 mm leaf discs werecut from the youngest leaves of transgenic plants approximately sixweeks after germination and 3 larvae (2nd stage) were allowed to feed oneach disc for 3-7 days. After 3-7 days the leaf discs were assessed forarea eaten, % of larval survivors and weight (in mg) of larvalsurvivors. Results of three separate experiments are shown in the tablesprovided below.

The results show elevated resistance to Heliothis in basic class IIIchitinase overexpressing plants when compared to non-transformed controllines. Heliothis virescens (the tobacco budworm) causes considerabledamage in tobacco crops and is particularly recalcitrant to controlusing pesticides as the larvae burrow deep within the plant.Furthermore, other Heliothis species cause similar damage to cotton andother crops.

% LEAF % SURV WT (mg) EATEN IVORS LARVAE (1) LARVAE Line Control 72 100 23 3505 C-15-5 45 92 14 3505 C-5-3 55 96 17 3505 C-4-2 57 92 19 3505C-12-8 — — — (2) LARVAE Line Control 77 100  26 3505 C-15-5 65 96 193505 C-5-3 60 92 23 3505 C-4-2 60 100  20 3505 C-12-8 44 87 15 (3) LineControl 65 100 53 3505 C-12-8 59 100 34 A summary of the two experimentsdetailed above is given below: (1) Line: Control 3505 C-15-5 3505C-5-3505 C-4-2 Resistance 0 4 2.5 2 Index (2) Line: Control 3505 C-15-53505 C-5-3505 C-4-2 3505 C-12-8 Resistance 0 2 3   3 4 Index (3) Line:Control 3505 C-12-8 Resistance 0 3 Index Resistance Index: S =significantly more susceptible than control plants (i.e. wild-type) (S)= more susceptible than control plants, but not significantly in allevaluations 0 = like wild type plants 1 = some signs of resistance butnot significantly different to controls 2 = identified resistance,significant only in some evaluations 3 = resistance with smallsignificant differences in all evaluations 4 = high resistance, butsymptoms or damage can still be observed 5 = total resistance. Nosymptoms or damage.

Example 167 Alternative Method for Introducing and Expressing More ThanOne Anti-Pathogenic Sequence In Plant Tissue

In addition to the possibility of expressing more than one transgene intransgenic lines by the sexual crossing of lines which are transgenicfor one gene only (as described above), the skilled artisan willrecognize that an equivalent way of generating lines transgenic for morethan one gene is by the use of transformation plasmids which carry morethan one gene. For example, the expression of two cDNAs in a transgenicline can be achieved by the transformation of the host plant with avector carrying each cDNA under the independent regulation of twopromoters i.e. the vector carries two expression cassettes in additionto sequences needed for antibiotic selection in vitro. Each expressioncassette can also carry any signal sequence, vacuolar targettingsequence and transciptional terminator so desired. Vectors carryingmultiple expression cassettes can be constructed for use withAgrobacterium transformation or direct gene transfer transformationsystems.

A further method for the expression of more than one transgene in atransgenic plant line is to firstly transform with a single gene (withappropriate regulatory signals) carried on a transformation vector andsubsequently transform a line selected from this transformation with afurther gene (with appropriate regulatory signals) carried on adifferent plasmid which utilizes a different antibiotic selectionsystem. This method is obvious to those of skill in the art.

Example 168 Synergistic Effect of Combined Anti-Pathogenic Sequences

Overexpression of two or more PR proteins in a transgenic plant givesrise to a synergistic anti-pathogen effect. The table below shows datafrom an experiment in which control and transgenic tobacco lines wereinoculated with the pathogen Peronospora tabacina, 5 and 8 days afterinoculation, percentage leaf area infected was assessed. The left sideof the table shows raw data from numerous lines of each phenotppe,whereas the right side shows mean data for each phenotype. In additionto the mean values for percentage leaf area infected, the relative areacompared as a percentage of the control (=100%) is presented. Thisenables a calculation of the expected value in the line expressing bothPR-1a and SAR8.2 assuming that the individual components are additive inaction. The observed values of 40.8 and 30.4% are well below theexpected values of 63.4 and 62.4% for 5 and 8 days post-inoculation andtherefore the disease resistance effects of PR-1a and SAR8.2 aresynergistic in transgenic plants.

TABLE A DAYS AF- MEAN DATA TER INOC- DAYS AFTER ULATION INOCULATION 5 85 8 Control (L6) 36.1 42.2 CONTROL 40.2 49.4 Control (L18) 42.8 51.1(100)    (100)    Control (L22) 41.7 55.0 PR-1a (L2) 35.0 42.8 PR.1a30.4 34.3 PR-1a (L7) 25.0 25.0 (75.6) (69.4) PR-1a (L13) 31.1 35.0SAR8.2 (L1) 36.7 49.4 SAR8.2 33.7 44.7 SAR8.2 (L8) 36.1 45.0 (83.8)(89.9) SAR8.2 (L11) 26.7 45.0 SAR8.2 (L21) 35.6 46.1 SAR8.2 (L25) 33.337.8 PR-1a/SAR8.2 (L17) 16.1 15.0 PR.1a/SAR8.2 16.1 15.0 (40.8) (30.4)Expected Additive Values: 63.4 62.4

Example 168A Expression of SAR/CHX-independent Genes in TransgenicPlants

The cDNAs described in examples 40A and 40B can be expressed intransgenic plants using techniques well known in the art. As componentsof the signal transduction pathway involved in SAR, the CHX-independentgenes are useful for the manipulation of the SAR response. For example,the constitutive expression of key components in the SAR transductionpathway in transgenic plants will likely lead to the generation ofplants with enhanced disease resistance characteristics and this willlikely be achieved by the activation of components in the pathwaydownstream to the component being expressed transgenically and hence tothe activation of anti-pathogenic end products. By way of illustrationthis may be achieved from the expression of the appropriate genes behindthe constitutive 35S promoter. cDNAs may be transferred to the vectorpCGN1761 or pCGN1761/ENX which carry the double 35S CaMV promoter andthe tml transcriptional terminator on a pUC-derived plasmid. Coloniescarrying the cDNA in sense are recovered and the cDNA carryingexpression cassette is subsequently excised and cloned into pCIB200 foruse in plant transformations using Agrobacterium. For direct genetransfer, the cDNA carrying expression cassette is transferred to thevector pCIB3064. Transformation to transgenic plants is undertaken usingtechniques well known in the art. For transformation of dicotyledonousspecies using binary Agrobactedrim vectors such as pCIB200 see Alexanderet al., Proc. Natl. Acad. Sci. 90: 7327-7331 (1993), and fortransformation of monocotyledonous species using direct gene transfervectors such as pCIB3064 see Koziel et al., Biotechnology 11: 194-200(1993). Transgenic plants are screened for high-level expression of theappropriate cDNA by Northern or Westem analysis. Plants which expresshigh levels of the gene product are found to have enhanced resistance toplant pathogens.

Other promoters are suitable for the expression of these cDNAs intransgenic plants. These include (but are not restricted to)constitutive promoters (such as those from the ubiquitin and actingenes) and cell and tissue-specific promoters.

For genes involved in the signal transduction of SAR which may causenegative regulation of the SAR pathway, increased disease resistance canbe achieved from the constitutive expression of cDNA in antisense to thegene coding sequence. The cloning and transfer of antisense sequences isundertaken in the same way as described above, except that theorientation of the cDNA is inverted to effect expression of antisensetranscripts.

M. Enhanced Chemical Regulation via Inactivation of EndogenousRegulation Example 169 Cloning of a DNA Fragment Encoding thePseudomonas putida NahG gene into the plant expression vector pCIB200

Genes encoding enzymes involved in the metabolic pathway which convertsnaphthalene to pyruvate and acetaldehyde in the bacterium Pseudomonasputida PpG7 are organized in two operons on the plasmid NAH7. Salicylatehydroxylase, which catalyzes the conversion of salicylate to catechol,is encoded by nahG. pSR20, a plasmid NAH subclone obtained from M.Schell (University of Georgia), was digested with SspI and HpaI toobtain a ca 1.5 kb restriction fragment containing nahG. This SspI-HpaIfragment was ligated to EcoRI linkers, digested with EcoRI, and clonedinto the EcoRI site of pCGN1761, a derivative of pCGN2113 (ATCC 40587)to add plant-recognized regulatory sequences.

pCGN1761 is prepared by digesting pCGN2113 with EcoRI and ligating theplasmid in the presence of a synthetic DNA adaptor containing an XbaIsite and a BamHI site. The adaptor contained EcoRI sticky ends on eitherend, but the adjacent bases were such that an EcoRI site was notreconstructed at this location. pCGN1761 contains a double CaMV 35Spromoter and the tml-3′ region with an ECoRI site between contained in apUC-derived plasmid backbone. The promoter-EcoRI-3′ processing sitecassette is bordered by multiple restriction sites for easy removal.

A pCGN1761 derivative was identified with the nahG gene oriented 5′ to3′ behind a double 35S promoter from the Cauliflower Mosaic Cirus, andfollowed by the efficient 3′ terminator tml. This plasmid was digestedwith XbaI to release a 4.7 kb restriction fragment containing the nahGconstruct, which was subsequendy cloned into the IbaI site of the planttransformation vector pCIB200. See, Uknes et al., Plant Cell 5: 159-169(1993).

Example 170 Transformation of the NahG Containing Plant ExpressionVector pCIB200/nahG into Nicotiana tobacum cv. Xanthi-nc

pCIB200/nahG was first transferred from E. coli to Agrobacteriumtumefaciens strain CIB542 (Uknes et al., supra) using theelectroporation method described by Wenjun and Forde, Nucleic Acids Res.17: 8385 (1989). Transformed Agrobacterium colonies were selected on 25ug/ml kanamycin.

Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacumcv Xanthi-nc was undertaken essentially as described by Horsch et al.,Science 227: 1229-1231 (1985). Leaf disks of Nicotiana tabacum cvXanthi-nc were infected with the Agrobacterium tumefaciens straincarrying pCIB200/nahG and selected for callus growth on kanamycin. Asingle shoot was regenerated (T1 generation) from each leaf disk andgrown in soil until seed set. Seed resulting from self-pollination (T2generation) of the regenerated transformants was scored for antibioticresistance on MS medium (Murashige and Skoog, Physiol. Plant: 473 (1962)containing 150 ug/ml kanamycin. Lines homozygous for the transgene wereidentified by allowing ten kanamycin resistant T2 progeny from eachindependent transformant to self-pollinate and set seed and screeningfor plants whose seed (T3 generation) were 100% kanamycin resistant.

Plants expressing nahG at high levels were not visually distinct fromwild-type plants. They flowered and set seed normally.

Example 171 Analysis of Transformants Expressing the NahG Gene

Idependently transformed plants were screened by RNA blot analysis(Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, Wiley &Sons, New York (1987)) for nahG MRNA accumulation and severaltransformants were selected (Nahg-1, -1, -2, -3, -8, -9 and -10). Theseplants were allowed to set seed and homoxygous T3 seed for furtheranalysis was generated as described above.

A. Effect of NahG Expression on the Accumulation of Salicylic Acid

To determine the effect of the expression of the nahG gene on theaccumulation of salicylic acid, several plants from each of the linesselected above were inoculated with TMV. After 7 days, when lesions hadformed on the infected leaves, leaf tissue was harvested and assayed fornahG MRNA, the salicylate hydroxylase protein, and salicylic acid.Assessment of mRNA abundance was undertaken by RNA blot analysis asdescribed above.

Salicylate hydroxylase protein was determined by Westem analysis usingstandard techniques. See, Pratt et al., Modern Methods Plant Anal. NewSeries 4: 51 (1986). Antiserum raised to salicylate hydroxylase waspurified from expression of the nahG gene in the E. coli expressionvector pGEX-2Tb.

Salicylic acid concentration was detemined after extraction from leaftissue using the technique essentially as described by Yalpani et al,Plant Cell 3: 809-818 (1991), except that samples were not allowed tooverdry, and that the final samples were resuspended in 500 ul of 20%methanol. 10-100 ul were injected onto a Dynamax 60A, 8 um, C-18 (4.6mm×25 cm) column with guard column (Rainin Instruments Co., Emeryville,Calif.) maintained at 40 C. Isocratin separation was performed at 1ml/min using 20% (v/v) methanol in 20 Mm sodium acetate, Ph 5.0.Fluorescence detection was done using a Model 980 detector (ABI/Kratosanalytical, Forster City, Calif.) with a 5 ul flowcell, deuterium lampwith a 295 nm excitation setting and a 370 nm cutoff emission filter.Thc limit of detection was 500 pg SA in 50 ul. Quantification wasdetermined versus a linear range (10-1000 ng/ml) of calibrationstandards for sodium salicylate.

The nahG-3, -8 and -10 lines expressed high levels of nahG mRNA andsalicylate hydroxylase protein. These lines accumulated about 100 ng/gSA following TMV treatment (see Table 1, below) which representedapproximately a 2-3 fold increase above the concentration inbuffer-treated control plants. Lines nahG-1 and -2 expressedintermediate levels of mRNA and barely detectable levels of salicylatehydroxylase protein, but accumulated 2824 and 979 ng/g SA, respectively,following TMV treatment, representing an 80 and a 30-fold induction,respectively. Line nahG-9 did not have detectable levels of either nahGmRNA or salicylate hydroxylase protein and accumulated 6334 ng/g SA.Similarly, the accumulation of SA in the non-transformed control linewas 5937 ng/g, representing a 180-fold induction.

These results showed a tight inverse correlation between expression ofthe nahG transgene and accumulation of SA. The presence of high levelsof nahG mRNA and salicylate hydroxylase protein resulted in asignificant block in SA accumulation in TMV-treated transgenic plants.

TABLE 1 Salicylic Acid Levels (ng/g tissue) ± Standard Deviation Xanthi32.1 ± 2.3 5937 ± 1011 185  NahG-1 35.8 ± 2.2 2824 ± 1461 79 NahG-2 38.8 ± 10.2 979 ± 113 25 NahG-3 41.3 ± 5.6 107 ± 45   3 NahG-8 36.2 ±2.7 81 ± 22  2 NahG-9 35.6 ± 3.0 6334 ± 765  179  NahG-10 33.9 ± 3.5 112± 4   3

All results were the average standard deviation, after the results werecorrected for recovery (57.1%). All values were based on triplicateassays, except for NahG/buffer, whose value was based on duplicateassay.

B. Effects of NahG Expression on Systemic Acquired Resistance (SAR)

To determine the effects of the reduced accumulation of SA on SAR,transgenic lines were challenge-inoculated with TMV. Lesion size wasscored 7 days later and compared to lesion size on buffer treatedcontrols (see Table 2, below). Control non-transgenic lines showed areduction of lesion size of 63% relative to buffer-treated plants, whichis typical of the SAR response to TMV. In the lines expressing highlevels of nahG mRNA and salicylate hydroxylasc protein, which were shownabove not to accumulate SA, lesion size was reduced by only 5-9%(nahG-3, -8 and -10). Lines expressing intermediate levels of nahG mRNAand salicylate hydroxylase protein showed an intemediate reduction inlesion size (nahG-1, and -2). The nahG-9 line, which did not expressdetectable levels of nahG mRNA or salicylate hydroxylase protein, showeda 66{circumflex over ( )} reduction in lesion size.

These results clearly demonstrate that SA is required for the onset ofSAR and is a cell signal in the SAR transduction pathway which can beeffectively eliminated.

TABLE 2 Lesion Size (Average Standard Deviation) LINE BUFFER n TMV n %REDUCTION Xanthi 3.5 ± 0.4(d) 3 1.3 ± 0.5(d) 5 63 NahG-1 4.1 ± 0.4(b) 32.7 ± 0.6(c) 5 34 NahG-2 4.4 ± 0.4(a) 3 3.8 ± 0.5(b) 5 14 MahG-3 4.4 ±0.4(a) 2 4.0 ± 0.5(b) 5  9 NahG-8 4.2 ± 0.4(b) 2 4.1 ± 0.7(b) 5  5NahG-9 3.8 ± 0.4(c) 3 1.3 ± 0.7(d) 5 66 NahG-10 4.5 ± 0.4(a) 3 4.2 ±0.8(a) 5  7

Three to five plants were analyzed per sample as indicated. Per plant,10 lesions were measured on 3 leaves. The date were analyzedstatistically by ANOVA II, followed by a Tukey-Kramer test. Within eachtreatment, statistically equivalent groups (p=0.05) are shown (a-d).

C. Benzo-1,2,3-thiodiazole-7-carboxylic Acid Indication of SAR in PlantsExpressing nahG

Three lines, nahG-3, -8 and -10, were shown above to express high levelsof nahG mRNA and salicylate hydroxylase protein, and to be blocked intheir SAR response to disease infection. Plants of these lines weretreated with the inducing chemical benzo-1,2,3-thiodiazole-7-carboxylicacid, and were shown to possess the SAR response. This resultdemonstrated the position of benzol-1,2,3-thiodiazole-7-carboxylic aciddownstream relative to SA in the SAR signal transduction pathway.

D. Chemical Regulaton of Gene Expression in NahG-expressing Plants

The PR-1a promoter (Uknes et al., Plant Cell 5: 159-169 (1993) ischemically regulated by exogenously appliedbenzo-1,2,3-thiodiazole-7-carboxylic acid and derivatives thereof, aswell as by SA. Plants possessing the PR-1a promoter fused to the GUSreporter gene are crossed to nahG-expressing lines nahG-3, -8 and -10.Progeny lines carrying both transgene constructions are found to expressGUS when induced by Benzo-1,2,3-thiodiazole-7-carboxylic acid, but notwhen treated with SA. Further, there is not GUS expression in responseto fluctuating endogenous levels of SA as would occur in plants notexpressing the nahG gene prior to and during flowering, for example.Consequently, the chemical regulation of the PR-1a gene promoter can beutilized without the activation of the endogenous cell signal SA.

E. Chemical Regulation of a Gene Encoding the Deka-endotoxin of Bacillusthurigensis in nahG-expressing Plants

Plants possessing the PR-1a promoter fused to a gene encoding thedelta-endotoxin of Bacillus thuringensis (Williams et al.,Bio/Technology 10: 540-543 (1992) are crossed to nahG-expressing linesnahG-3, -8 and -10. Progeny lines carrying both transgene constructionsare found to express the endotoxin gene when induced bybenzo-1,2,3-thiodiazole-7-carboxylic acid, but not when treated with SA.Further, there is no endotoxin gene expression in response tofluctuating endogenous levels of SA as would occur in plants notexpressing the nahG gene prior to and during flowering, for example.Consequently, the chemical regulation of the PR-1a gene promoter can beutilized without the activation of the endogenous cell signal SA.

N. EXPLOITATION OF DISEASE SUSCEPTIBILITY CREATED BY INACTIVATION OFENDOGENOUS REGULATION

Apart from their utility in chemical regulation, plants which aredisrupted in the signal transduction cascade leading to the expressionof PR-proteins and therefore systemic acquired resistance, have furtherutility for disease testing. Plants incapable of expressing PR proteinsdo not develop the systemic acquired resistance response and thusdevelop larger lesions more quickly when challenged by pathogens. Theseplants are useful as “universal disease susceptible” (UDS) plants byvirtue of their being susceptible to many strains and pathotypes ofpathogens of the host plant and also to pathogens which do not normallyinfect the host plant, but which infect other hosts. They provide usefulindicators of evaluation of disease pressure in field pathogenesis testswhere the natural resistance phenotype of so-called wild-type (i.e.non-transgenic) plants may vary and therefore not provide a reliablestandard of susceptibility. Furthermore, these plants have additionalutility for the the testing of candidate disease resistance transgenes.Using a nahG-expressing stock line as a recipient for transgenes, thecontribution of the transgene to disease resistance is directlyassessable over a base level of susceptibility. A further utility ofnahG-expressing plants is as a tool in the understanding ofplant-pathogen interactions. NahG-expressing host plants do not mount asystemic response to pathogen attack, and the unabated development ofthe pathogen is an ideal system in which to study its biologicalinteraction with the host. As nahG-expressing host plants may also besusceptible to pathogens outside of whose host-range they normally fall,these plants also have significant utility in the molecular, genetic,and biological study of host-pathogen interactions. Furthermore, the UDSphenotype of nahG-expressing plants also renders them of utility forfungicide screening. Plants expressing nahG in a particular host haveconsiderable utility for the screening of fungicides using that host andpathogens of the host. The advantage lies in the UDS phenotype of thenahG-expressing host which circumvents the problems encountered by hostsbeing differentially susceptible to different pathogens and pathotypes,or even resistant to some pathogens or pathotypes. nahG-expressingplants have further utility for the screening of fungicides against arange of pathogens and pathotypes using a heterologous host i.e. a hostwhich may not normally be within the host species range of particularpathogens. Thus, the susceptibility of nahG-expressing host plants suchas Arabidopsis, which are easily manipulable and have limited spacerequirements, to pathogens of other species (e.g. crop plant species)would facilitate efficacious fungicide screening procedures forcompounds against important pathogens of crop plants.

In the situations described above the nahG gene can be expressed behindpromoters which are expressible in plants using gene cloning techniqueswhich are well known in the art. A preferred promoter would express nahGat the time of disease challenge. A particularly preferred promoterwould be expressed constitutively e.g. the Cauliflower Mosaic Virus 35Spromoter.

Example 172 Use of NahG-Expressing Plant Lines in Disease Testing

Tobacco and Arabidopsis plants expressing the nahG gene constitutivelywere challenged with numerous pathogens and found to develop largerlesions more quickly than wild-type plants. This phenotype is referredto as UDS (i.e. universal disease susceptibility) and is a result of theplants being unable to express SAR genes to effect tie plant defenceagainst pathogens by virtue of the expression of salicylate hydroxylaseprotein, and the inactivation of salicylic acid as an endogenous cellsignal in systemic acquired resistance. Table 2 (in example 5) shows acomparison of lesion size in plants expressing nahG and wild-type plantsand table 6 (below) shows a comparison of the development of lesionsover an eight-day period in wild-type tobacco and nahG-expressingtobacco.

TABLE 3 Lesion Growth Over a Period of Eight Days in NahG-transgenic andNon-transgenic Tobacco Xanthi NahG-10 mean SD mean SD DPI  50.8 12.4 47.5 12.6 2 102.2 19.3 113.4 26.7 3 138.0 25.6 186.4 29.0 4 170.8 29.7227.3 27.1 5 199.0 34.7 276.5 35.3 6 220.9 39.8 332.4 38.0 7 234.2 47.5376.4 42.5 8

Plants of nahG-expressing line NahG-10 (see tables 1 and 2) andwild-type tobacco cultivar Xanthi were inoculated with TMV and lesionsize was monitored over a period of 8 days post inoculation (DPI). Meanvalues for lesion size are reported in {fraction (1/1000)} inch withstandard deviation (SD). The nahG-expressing line develops largerlesions more quickly than does wild-type Xanthi.

The UDS phenotype of these nahG expressing plants renders them useful ascontrol plants for the evaluation of disease symptoms in experimentallines in field pathogenesis tests where the natural resistance phenotypeof so-called wild-type lines may vary (i.e. to different pathogens anddifferent pathotypes of the same pathogen). Thus, in a field environmentwhere natural infection by pathogens is being relied upon to assess theresistance of experimental lines, the incorporation into the experimentof nahG expressing lines of the appropriate crop plant species wouldenable an assessment of the true level and spectrum of pathogenpressure, without the variation inherent in the use of non-experimentallines.

Example 173 Assessment of the Utility of Transgenes for the Purposes ofDisease Resistace

Plants constitutively expressing nahG are used as host plants for thetransformation of transgenes to facilitate their assessment for use indisease resistance. A stock of Arabidopsis or tobacco plants is createdwhich express the nahG gene. This stock is used for subsequenttrasformations with candidate genes for disease resistance thus enablingan assessment of the contribution of an individual gene to resistanceagainst the basal level of the UDS nahG expressing plants.

Example 174 NahG-expressing Plants as a Tool in UnderstandingPlant-Pathogen Interactions

Plants expressing nahG are useful for the understanding of plantpathogen interactions, and in particular for the understanding of theprocesses utilized by the pathogen for the invasion of plant cells. Thisis so because nahG-expressing host plants do not mount a systemicresponse to pathogen attack, and the unabated development of thepathogen is an ideal scenario in which to study its biologicalinteraction with the host.

Of futher significance is the observation that a host species expressingnahG may be susceptible to pathogens not normally associated with thatparticular host, but instead associated with a different host.Arabidopsis plants were transformed with nahG and those expressing thegene were characterized by the UDS phenotype. These plants arechallenged with a number of pathogens which normally only infecttobacco, and found to be susceptible. Thus, the expression of nahG in ahost plant and the accompanying UDS phenotppe leads to a modification ofpathogen-range susceptibility and this has significant utility in themolecular, genetic amd biochemical analysis of host-pathogeninteraction.

Example 175 NahG-expressing Plants for Use in Fungicide Screening

Plants expressing nahG are particularly useful in the screening of newchemical compounds for fungicide activity. The advantage lies in the UDSphenoytpe of the nahG-expressing host plant which circumvents theproblems encountered by the host being differentially susceptible todifferent pathogens and pathotypes, or even resistant to some pathogensor pathotypes. By way of example transgenic wheat expressing nahG couldbe effectively used to screen for fungicides to a wide range of wheatpathogens and pathotypes as the nahG-expressing line would not mount aresistance response to the introduced pathogen and would not displaydifferential resistance to different pathotypes which might otherwiserequire the use of multiple wheat lines, each adequately susceptible toa particular test pathogen. Wheat pathogens of particular interestinclude (but are not limited to) Erisyphe gaminis (the causative agentof powdery mildew), Rhizoctonia solani (the causative agent of sharpeyespot), Pseudocercosporella herpotrichoides (the causative agent ofeyespot), Puccinia spp. (the causative agents of rusts), and Septorianodorum. Similarly, corn plants or tobacco plants expressing nahG wouldbe highly susceptible to their respective pathogens and would thereforebe useful in the screening for fungicides.

nahG-expressing plants have further utility for the screening of a widerange of pathogens and pathotypes in a heterologous host i.e. in a hostwhich may not normally be within the host species range of a particularpathogen and which may be particularly easily to manipulate (such asArabidopsis). By virtue of its UDS phenotype the heterologous hostexpressing nahG is susceptible to pathogens of other plant species,including economically important crop plant species. Thus, by way ofexample, the same Arabidopsis nahG-expressing line could be infectedwith a wheat pathogen such as Erisyphe graminis (the causative agent ofpowdery mildew) or a corn pathogen such as Helminthosporium maydis andused to test the efficacy of fungicide candidates. Such an approach hasconsiderable improvements in efficiency over currently used proceduresof screening individual crop plant species and different cultivars ofspecies with different pathogens and pathotypes which may bedifferentially virulent on the different crop plant cultivars.Furthermore, the use of Arabidopsis has advantages because of its smallsize and the possibility of thereby undertaking more tests with limitedresources of space.

While the present invention has been described with reference tospecific embodiments thereof, it will be appreciated that numerousvariations, modifications, and embodiments are possible, andaccordingly, all such variations, modifications and embodiments are tobe regarded as being within the spirit and scope of the presentinvention.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 111(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2038 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 932..1435 (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 1: CTCGAGGATT TCAAACTCTA GCTTCACTAA AACTTGAGCT TTCTTTTCCA CTAATGTCGA60 AAAACGAAAT AAACATAAGC TATTTACAAA AAATAAAAAA ATACTCCATT TGAATCTAAA 120GTCAAGTCGT GATTGGGATA AGAAAATAGA AATTTATTTA TACTCCAGAT CAAGCCGTGA 180TTGGAATGAG ATAATAGAAA AGTATGATAG TACATGAGTA ACATCAAGTT GGAAATTAAG 240GGAAGGAAAT TAGAGAAAGA ACTGAAGAAT ATCCAAATAT TCTTTACGTC CAAATTTGAT 300AGTTATTTAA CGTCATCGAG ATGACGGCCA TGTTCAAGTT TTCCACAAAT ATTGAGAAAA 360GAAAGAAGAA GACACAAACT GTGTTTGGTA TTATTATAGT TTTTTCTTTT AGAGAATTGA 420TTGTACATAT AAGAAATATA ATATAAGATT TAGAAATAAG ATTATTAGAA AAATCAAACA 480TCAAAGTATT TATTTTAAAT TCTTTTTCCA ATGGACATTC CCATTCTGAA AAAAAAGAGA 540TATAAATATG GAAGTAAAAA TTAATCAGAT CGTTAAATGT AGAAAATATT AATTAACACA 600TTAACCATAA CCAGTCTACT TTATTTAACA AAAAGCACAT CTGATAGATC AAAAAAGTGT 660TTAACTTCAT GCATTGACAA TTTAAAATTA TTTTGCAACA TCGGGTAAAA CTATTTTACA 720ACAATTGGTA ACTGCATATA TAAGTTTAAT ATGGTAACCT AGAAAATAGG ATAAATTATC 780TATAACAGGA TATATTACAT TGATATTACC ATGTCAAAAA ATTTAGTAAG TACATGAATA 840ATCACCGTGA AATCTTCAAG ATTTCTCCTA TAAATACCCT TGGTAGTAAA TCTAGTTTTT 900CCATTCAAGA TACAACATTT CTCCTATAGT C ATG GGA TTT GTT CTC TTT TCA 952 MetGly Phe Val Leu Phe Ser 1 5 CAA TTG CCT TCA TTT CTT CTT GTC TCT ACA CTTCTC TTA TTC CTA GTA 1000 Gln Leu Pro Ser Phe Leu Leu Val Ser Thr Leu LeuLeu Phe Leu Val 10 15 20 ATA TCC CAC TCT TGC CGT GCC CAA AAT TCT CAA CAAGAC TAT TTG GAT 1048 Ile Ser His Ser Cys Arg Ala Gln Asn Ser Gln Gln AspTyr Leu Asp 25 30 35 GCC CAT AAC ACA GCT CGT GCA GAT GTA GGT GTA GAA CCTTTG ACC TGG 1096 Ala His Asn Thr Ala Arg Ala Asp Val Gly Val Glu Pro LeuThr Trp 40 45 50 55 GAC GAC CAG GTA GCA GCC TAT GCG CAA AAT TAT GCT TCCCAA TTG GCT 1144 Asp Asp Gln Val Ala Ala Tyr Ala Gln Asn Tyr Ala Ser GlnLeu Ala 60 65 70 GCA GAT TGT AAC CTC GTA CAT TCT CAT GGT CAA TAC GGC GAAAAC CTA 1192 Ala Asp Cys Asn Leu Val His Ser His Gly Gln Tyr Gly Glu AsnLeu 75 80 85 GCT GAG GGA AGT GGC GAT TTC ATG ACG GCT GCT AAG GCT GTT GAGATG 1240 Ala Glu Gly Ser Gly Asp Phe Met Thr Ala Ala Lys Ala Val Glu Met90 95 100 TGG GTC GAT GAG AAA CAG TAT TAT GAC CAT GAC TCA AAT ACT TGTGCA 1288 Trp Val Asp Glu Lys Gln Tyr Tyr Asp His Asp Ser Asn Thr Cys Ala105 110 115 CAA GGA CAG GTG TGT GGA CAC TAT ACT CAG GTG GTT TGG CGT AACTCG 1336 Gln Gly Gln Val Cys Gly His Tyr Thr Gln Val Val Trp Arg Asn Ser120 125 130 135 GTT CGT GTT GGA TGT GCT AGG GTT CAG TGT AAC AAT GGA GGATAT GTT 1384 Val Arg Val Gly Cys Ala Arg Val Gln Cys Asn Asn Gly Gly TyrVal 140 145 150 GTC TCT TGC AAC TAT GAT CCT CCA GGT AAT TAT AGA GGC GAAAGT CCA 1432 Val Ser Cys Asn Tyr Asp Pro Pro Gly Asn Tyr Arg Gly Glu SerPro 155 160 165 TAC TAATTGAAAC GACCTACGTC CATTTCACGT TAATATGTATGGATTGTTCT 1485 Tyr GCTTGATATC AAGAACTTAA ATAATTGCTC TAAAAAGCAACTTAAAGTCA AGTATATAGT 1545 AATAGTACTA TATTTGTAAT CCTCTGAAGT GGATCTATAAAAAGACCAAG TGGTCATAAT 1605 TAAGGGGAAA AATATGAGTT GATGATCAGC TTGATGTATGATCTGATATT ATTATGAACA 1665 CTTTTGTACT CATACGAATC ATGTGTTGAT GGTCTAGCTACTTGCGATAT TACGAGCAAA 1725 ATTCTTAACT ACATGCCTTA GGAACAAGCT TACACAGTTCATATAATCTA CTAGAGGGCC 1785 AAAAACATGA AAATTACCAA TTTAGATGGT AGGAGGATATTGAAAGTGGA GCAGCTAGTT 1845 TTAATAACTG ACCGTTAGTC TTAAAATTGA CGGTATAAAAATATTTACAT AATCAGGTCA 1905 TTTATAAGGT AATTATAGGT AAATATTTAT GACGAATTCTCAATAGTAAT CTGAAAAAAA 1965 ATTGTAACTA ACCTATTATA CTAAAACTAC TATAATAGGTTAGATTACAT TAATCATGTC 2025 ATTAGAAGAT CTT 2038 (2) INFORMATION FOR SEQID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2256 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:716..1246 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTACTGTTAA AGTATTCGTACTCGTAGATA TTTCAAAAAT TAGATAGCGA TTTTATCATC 60 TAATCAACTA TAGATCTAAGTCGAAAATAT ACCTTGAGTA TAGATTTTTA CTTACCAGCC 120 TTTCATCTTC TTCCAGAAATAAGAGTGGAA ATCAACGGTA GCAGATAACG TTGAGATATG 180 ATCTTGTAGT AATTGACACCTAACGGGACT GCTTTCCTTT AAAATATAGA CGATAATATC 240 AGTTATAAGG ATGCTTTCACTTTCTAATTA AAGCATATTC TGATTCGGTT TTATAGAATT 300 TGATAGAAGT TAGACAGCATCCCTCCAATT GAACAGGTTC GATTACTCAA CTTCCCGTCT 360 ACTTAAACAA ATTCAACTCTTTTCGCGTAC TATATTTATG TTAAAATACA CTGGTAATAA 420 TGTATAAAAC TATATGTATGTATATAATAT AACTTCATGA TTTATCAAAG TTGGTCAAAT 480 TATGATCATG GCTATAATAGTTGTTCATCA ATAGATAGAA TTTTTTAATG GCGGCTTTTG 540 TTTCTTTAAC GAATATGATCTAACGTTCTT TGAATTAACG GAGCTATAAT TTTATACTAT 600 TCTTTTCAAA TATGGCTAAAGAGTTTGCTA ACAAGTTGCA AACTTTGTAA CTCAGCTATA 660 TTCTTCCCTA TAAAAATCATCCTTACTATG CTTTGTTTCT CACCAAAACA CAAAA ATG 718 Met 1 GGA TTC TTA ACA ACAATA GTT GCT TGT TTC ATT ACC TTT GCA ATA TTA 766 Gly Phe Leu Thr Thr IleVal Ala Cys Phe Ile Thr Phe Ala Ile Leu 5 10 15 ATT CAC TCA TCC AAA GCTCAA AAC TCC CCC CAA GAT TAT CTT AAC CCT 814 Ile His Ser Ser Lys Ala GlnAsn Ser Pro Gln Asp Tyr Leu Asn Pro 20 25 30 CAC AAT GCA GCT CGT AGA CAAGTT GGT GTT GGC CCC ATG ACA TGG GAC 862 His Asn Ala Ala Arg Arg Gln ValGly Val Gly Pro Met Thr Trp Asp 35 40 45 AAT AGG CTA GCA GCC TAT GCC CAAAAT TAT GCC AAT CAA AGA ATT GGT 910 Asn Arg Leu Ala Ala Tyr Ala Gln AsnTyr Ala Asn Gln Arg Ile Gly 50 55 60 65 GAC TGC GGG ATG ATC CAC TCT CATGGC CCT TAC GGC GAA AAC CTA GCC 958 Asp Cys Gly Met Ile His Ser His GlyPro Tyr Gly Glu Asn Leu Ala 70 75 80 GCC GCC TTC CCT CAA CTT AAC GCT GCTGGT GCT GTA AAA ATG TGG GTC 1006 Ala Ala Phe Pro Gln Leu Asn Ala Ala GlyAla Val Lys Met Trp Val 85 90 95 GAT GAG AAG CGT TTC TAT GAT TAC AAT TCAAAT TCT TGT GTA GGA GGA 1054 Asp Glu Lys Arg Phe Tyr Asp Tyr Asn Ser AsnSer Cys Val Gly Gly 100 105 110 GTA TGT GGA CAC TAT ACT CAG GTG GTG TGGCGT AAC TCA GTA CGT CTC 1102 Val Cys Gly His Tyr Thr Gln Val Val Trp ArgAsn Ser Val Arg Leu 115 120 125 GGT TGT GCT AGG GTT CGA AGC AAC AAT GGTTGG TTT TTC ATA ACT TGC 1150 Gly Cys Ala Arg Val Arg Ser Asn Asn Gly TrpPhe Phe Ile Thr Cys 130 135 140 145 AAT TAT GAT CCA CCA GGT AAT TTT ATAGGA CAA CGT CCC TTT GGC GAT 1198 Asn Tyr Asp Pro Pro Gly Asn Phe Ile GlyGln Arg Pro Phe Gly Asp 150 155 160 CTT GAG GAG CAA CCC TTT GAT TCC AAATTG GAA CTT CCA ACT GAT GTC 1246 Leu Glu Glu Gln Pro Phe Asp Ser Lys LeuGlu Leu Pro Thr Asp Val 165 170 175 TAATGAGTGC ACGTACATGA ACAAATTATCATGAATAAAG GAAAATAAAA TGCAGTGCTA 1306 TGCTATGTGA TTTTAGCTTC CCAGTTGGATAATAATCTGA TGGTGTAGTA ACTGGCCGAG 1366 TGTTTGGACC CTACCTTGCA TGTTGGTAGATGGAATCAGT TTATATATGA GTTTTCTGTT 1426 GGCTTATGTT ATTTTTATTA AAATCTTATTCTAGTTTTAC AGTTTTTTAT TCGCACTATA 1486 TGTGTATGGG TCCAAATTTG ATCGACCACGACCTATGACG AGTACAACAA ACGACCGGGT 1546 ACCTTCGAAT CGGCGTCCCA AGGGAAACGACCCTAGGGCG AAAGAAGAGA CTGTATGACT 1606 CTTGTAGTAG TGTAAGCCCA TTAGGGAACATTAAGAATAT TCCGCCGAAT ACTAATTGTA 1666 TTTTGTTTCT TACAATTCGG AAGGGTTTCTCTCTAGTATA TAAAGGGGAC AAAAAACCTT 1726 GTAAGTGGCG GTGGATACTC TGAGAAGCTTACTAATCTTG GATGAAAAGA AAAACTCTCT 1786 TCTCTTTATC TATTATTATT CTAGAGATTATTATCTTCAG CTACGATTTA CCCCTTCATC 1846 TTTGATTGAT TTGTTCAAAA AGGTTTTAACATCTTTTGAG TCAAACAATT TGGCNCCGTC 1906 TATGGGGATT TCTATAGCTG AAATCATAGTTCTCATCTAG ATTTCTGAAG TGATAATTAC 1966 TTTTCTTCAA ACCTCATAAA AATCAACAATGGCSGGAAAG GAAGTGAAGC TAAAGGCGGT 2026 ASAGATCGTC TCGAATAACT CCTGAACTCCCTCAACGAAG ACGGCAGAGA AGACAATGAG 2086 AATACAACAC CAAGCGCTAC ACCGGAGGGAAGATCTCACC TCTTCCACAC GGGGATCTAA 2146 CGATCTTGCG CGAAAGGGGA GTCTCGACATCCACAGCAGG GGAAGCACCA CCAACACTCA 2206 AAAGGTTACT AGAATAATGG TTAACGAGTGCTTGGAGCAA CATGCTTGAG 2256 (2) INFORMATION FOR SEQ ID NO: 3: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1103 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAAGAAAGCT CTTTAAGCAATGGCTGCCCA CAAAATAACT ACAACCCTTT CCATCTTCTT 60 CCTCCTTTCC TCTATTTTCCGCTCTTCCGA CGCGGCTGGA ATCGCCATCT ATTGGGGTCA 120 AAACGGCAAC GAGGGCTCTCTTGCATCCAC CTGCGCAACT GGAAACTACG AGTTCGTCAA 180 CATAGCATTT CTCTCATCCTTTGGCAGCGG TCAAGCTCCA GTTCTCAACC TTGCTGGTCA 240 CTGCAACCCT GACAACAACGGTTGCGCTTT TTTGAGCGAC GAAATAAACT CTTGCAAAAG 300 TCAAAATGTC AAGGTCCTCCTCTCTATCGG TGGTGGCGCG GGGAGTTATT CACTCTCCTC 360 CGCCGACGAT GCGAAACAAGTCGCAAACTT CATTTGGAAC AGCTACCTTG GCGGGCAGTC 420 GGATTCCAGG CCACTTGGCGCTGCGGTTTT GGATGGCGTT GATTTCGATA TCGAGTCTGG 480 CTCGGGCCAG TTCTGGGACGTACTAGCTCA GGAGCTAAAG AATTTTGGAC AAGTCATTTT 540 ATCTGCCGCG CCGCAGTGTCCAATACCAGA CGCTCACCTA GACGCCGCGA TCAAAACTGG 600 ACTGTTCGAT TCCGTTTGGGTTCAATTCTA CAACAACCCG CCATGCATGT TTGCAGATAA 660 CGCGGACAAT CTCCTGAGTTCATGGAATCA GTGGACGGCG TTTCCGACAT CGAAGCTTTA 720 CATGGGATTG CCAGCGGCACGGGAGGCAGC GCCGAGCGGG GGATTTATTC CGGCGGATGT 780 GCTTATTTCT CAAGTTCTTCCAACCATTAA AGCTTCTTCC AACTATGGAG GAGTGATGTT 840 ATGGAGTAAG GCGTTTGACAATGGCTACAG CGATTCCATT AAAGGCAGCA TCGGCTGAAG 900 GAAGCTCCTA AGTTTAATTTTAATTAAAGC TATGAATAAA CTCCAAAGTA TTATAATAAT 960 TAAAAAGTGA GACTTCATCTTCTCCATTTA GTCTCATATT AAATTAGTGT GATGCAATAA 1020 TTAATATCCT TTTTTTCATTACTATACTAC CAATGTTTTA GAATTGAAAA GTTGATGTCA 1080 ATAAAAACAT TCCAAGTTTATTT 1103 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 900 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: AAAAAGAAAA AAAAAATGAA CTTCCTCAAA AGCTTCCCCTTTTTTGCCTT CCTTTATTTT 60 GGCCAATACT TTGTAGCTGT TACTCATGCT GCCACTTTTGACATTGTCAA CAAATGCACC 120 TACACAGTCT GGGCCGCGGC CTCTCCAGGT GGAGGCAGGCGGCTCGACTC AGGCCAATCT 180 TGGAGCATTA ATGTGAACCC AGGAACAGTC CAGGCTCGCATTTGGGGTCG AACCAATTGC 240 AACTTCGATG GCAGTGGCCG AGGTAATTGT GAGACTGGAGACTGTAACGG GATGTTAGAG 300 TGTCAAGGCT ATGGAAAAGC ACCTAACACT TTAGCTGAATTTGCACTTAA TCAACCCAAT 360 CAGGACTTTG TCGACATCTC TCTTGTTGAT GGATTTAACATCCCCATGGA ATTCAGCCCG 420 ACCAATGGAG GATGTCGTAA TCTCAGATGC ACAGCACCTATTAACGAACA ATGCCCAGCA 480 CAGTTGAAAA CACAAGGTGG ATGTAACAAC CCATGTACTGTGATAAAAAC CAATGAATAT 540 TGTTGTACAA ATGGGCCTGG ATCATGTGGG CCTACTGATTTGTCGAGATT TTTTAAGGAA 600 AGATGCCCTG ATGCTTATAG TTATCCACAG GATGATCCAACCAGTTTGTT TACGTGTCCT 660 TCTGGTACTA ATTACAGGGT TGTCTTCTGC CCTTGAAATTGAAGCCTGCA AAATTATGAC 720 TATGTAATTT GTAGTTTCAA ATATATAAGC TACACAAGTAGTACTAAGCA CTATTAAATA 780 AAAAAGAGAG TGACAAAGAG GAGAGGCTGT GGGTCAGATTCTCTTGTTCG CTGTTGTCGT 840 TGTTGTAGCA TTCTGGTTTT AAGAAATAAA GAAGATATATATCTGCTAAA TTATTAAATG 900 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4483 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAGCTCCTCT AGGGGGCCAA GGCAAACCTTTTTGCTAATG GGAAAAAAAT ATTAGACCAA 60 GAGTTTGTAA TAGCTACTCA ATTTCAATTTACAAAGGGGA AAATTTAACT GTTTTGCCCT 120 TATATCTTTT GGTCCCGAAA CATAAAATATCCCATCCGAA ATTCCAAATG GTCCATTATC 180 GGCAAGTAGC TTTCTTTTAT TATAGTTAGTTGACAAAACA CTATCGAGAT ATCATTAGTA 240 TAATAATAAC TTCAAAGTCC ATCTTCTTAGCTGCCTCCTC ATTAGAGCCG CCACTAAAAT 300 AAGACCGATC AAATAAAAGC CGCCATTAAAATAATGAATT TTAGGACTCT CAATTGTCAC 360 GTAAGTGCCA AAACTCTTCC AATACTTTGCTGCAACTTGG GGCTGCTAGC TTCTGAGCTT 420 CCTGGGATAT TTCTATGTTT ATCTCTTAATTTACATCTCA ACTAATATCA AGAAATTAAA 480 CAGGTGCAGC AAATCATAAA ATTTTCCTCTAAAGAAGAAA ATGACTCCGG TTACTGATTC 540 ATTGGCCTTT TCAGAGTCTG CGTGCCATATTCACTAATGG GTCGTTTGGT ACAAGAAATA 600 ATGATAATAA TTTCGAAATA GAATTTGGGATTTCATTTAT TTCATATTTA ATTATAAATA 660 TTAGCTAATT TCGAAATAAA TTTTACATTAAAATAGTGAA ATCAACTATC TCATATATAA 720 GGTGGAATAG CTAATCTCAT AGCCACCTCAGTTAGAATCC AGTTTCCTCT AATAAATGCA 780 GCGAAATATT AGAAGACTTT CATTAAATCAATTCATATAA TTTAAAAATA CTAGACATGG 840 AAAAAAAAAA CGATTCGAGA CTGTTATGGAAGGCGTTGCC TTCGATGTAG ATTCTCATCC 900 ATTGCTTTCG TGCAATAGCA ATATGACATCTTATTCTTAG AACTACTTTT AAATGAAAGT 960 CATATAGAAC TTTAAAATCT CTCAACTAGTTTTAAGGGAA TTCAAAATAC GACCAATATT 1020 TATTACTTAC TTATGGATAA ATTTAAATAATATGTATTTT ATCTTGAAAT TGAATTGAAA 1080 ATATTAAATT ACTTGGTTTA ATATAAACAATAGATATCGT TAAGTATTTA CCACAAACAT 1140 TTTATTAGTT GTAACGATGA TTAAGCAGGAATTCCTCTGG TTGTGCAGGA TGAAAGAAAC 1200 TAACTAGCTA TAATTTCTTT TGTAAAGTCAAGATAGTACG GCACCTTATG GAGAAAATAA 1260 ATAACTTTAC ATCATCAAAC TCATCTTCTTTTTTCCACAA AATGATCAAG TTGACATGTT 1320 AATAGCCAGG TCACCGGGGG CGGCTCTTAACTTCATTAGC CTACGAATAA CAAATCCAAT 1380 ATTATATTTA CACAAGGCTA TATATATATCTCAATATAAA TAGCTCGTTG TTCATCTTAA 1440 TTCTCCCAAC AAGTCTTCCC ATCATGTCTACCTCACATAA ACATAATACT CCTCAAATGG 1500 CTGCTATCAC ACTCCTAGGA TTACTACTTGTTGCCAGCAG CATTGACATA GCAGGTTTCT 1560 GGTCAAATAT TTGAACTTCC CAGCCAAAAATATTGTCTTA TAATTTTGTG TGCGCAAAAT 1620 TTTAATTTAG TTGATAGTTA TTTGCTTATTTTTCTTTTCA AATTGCTTGT GTTTTTTTCT 1680 CAAATTAACT TGCACCGTAT TCATTTAGCGATAGTTATTT GCTCTATTTT TGTGTAACAC 1740 TCACTCACAA ACTTTTCAAT TTGAGGGGAGGACAGTGAAT CTAAGATTGA AATTTATGAG 1800 TTTAATTAGA CTAATTCCCA TTTGATTTATTGGCTAGAAG TCAATTATTT GCATAGTGAG 1860 TCTTTTAACA CACAGATTTG AGTTAAAGCTACTACGTTCG TATTAACCCA TAACATATAC 1920 ACCTTCTGTT CTAATTTCTT TGACACTTTTTGTTAGTTTG TTCCAAAAAG GACGGACATA 1980 TTTGATATTT GAGAATACTT TACCTTAACCTTAATAGAAT TTTTTATGAC ATCACATATA 2040 TTATGGAATA TATACGACCA TAATTTTCAAATATCTTATA GTCGTACAAA TATTATAGCA 2100 TGTTTAATAC CACAACTTTC AAATTCTTCTTTTCCTTAAA AACAAAATAT GTCACATAAA 2160 TTAAAATAGA GGAAGTATAC TACATCAATCAGCCCCTAGT GGAGGGGACC CTACTGTAAG 2220 TTTTTAAGTT TTCAAGAATT CAGTAATTGATTAGGAGCCC GTCTGGACAT AAAAAAAAAT 2280 TCCTTTTTTT CCAAAAAATG CCCACTAAATTTCTAACACT ATTTTGTAAT TCTTATTGAG 2340 CAGGGGGCTC AATCGATAGG TGTTTGCTATGGAATGCTAG GCAACAACTT GCCAAATCAT 2400 TGGGAAGTTA TACAGCTCTA CAAGTCAAGAAACATAGGAA GACTGAGGCT TTATGATCCA 2460 AATCATGGAG CTTTACAAGC ATTAAAAGGCTCAAATATTG AAGTTATGTT AGGACTTCCC 2520 AATTCAGATG TGAAGCACAT TGCTTCCGGAATGGAACATG CAAGATGGTG GGTACAGAAA 2580 AATGTTAAAG ATTTCTGGCC AGATGTTAAGATTAAGTATA TTGCTGTTGG GAATGAAATC 2640 AGCCCTGTCA CTGGCACATC TTACCTAACCTCATTTCTTA CTCCTGCTAT GGTAAATATT 2700 TACAAAGCAA TTGGTGAAGC TGGTTTGGGAAACAACATCA AGGTCTCAAC TTCTGTAGAC 2760 ATGACCTTGA TTGGAAACTC TTATCCACCATCACAGGGTT CGTTTAGGAA CGATGCTAGG 2820 TGGTTTGTTG ATCCCATTGT TGGCTTCTTAAGGGACACAC GTGCACCTTT ACTCGTTAAC 2880 ATTTACCCCT ATTTCAGTTA TTCTGGTAATCCAGGCCAGA TTTCTCTCCC CTATTCTCTT 2940 TTTACAGCAC CAAATGTGGT GGTACAAGATGGTTCCCGCC AATATAGGAA CTTATTTGAT 3000 GCAATGCTGG ATTCTGTGTA TGCTGCCCTCGAGCGATCAG GAGGGGCATC TGTAGGGATT 3060 GTTGTGTCCG AGAGTGGCTG GCCATCTGCTGGTGCATTTG GAGCCACATA TGACAATGCA 3120 GCAACTTACT TGAGGAACTT AATTCAACACGCTAAAGAGG GTAGCCCAAG AAAGCCTGGA 3180 CCTATTGAGA CCTATATATT TGCCATGTTTGATGAGAACA ACAAGAACCC TGAACTGGAG 3240 AAACATTTTG GATTGTTTTC CCCCAACAAGCAGCCCAAAT ATAATATCAA CTTTGGGGTC 3300 TCTGGTGGAG TTTGGGACAG TTCAGTTGAAACTAATGCTA CTGCTTCTCT CGTAAGTGAG 3360 ATGTGAGCTG ATGAGACACT TGAAATCTCTTTACATACGT ATTCCTTGGA TGGAAAACCT 3420 AGTAAAAACA AGAGAAATTT TTTCTTTATGCAAGATACTA AATAACATTG CATGTCTCTG 3480 TAAGTCCTCA TGGATTGTTA TCCAGTGACGATGCAACTCT GAGTGGTTTT AAATTCCTTT 3540 TCTTTGTGAT ATTGGTAATT TGGCAAGAAACTTTCTGTAA GTTTGTGAAT TTCATGCATC 3600 ATTAATTATA CATCAGTTCC ATGTTTGATCAGATTGGGAT TTGGTAACTT CAATGTTTAG 3660 TTATTTATTA ATTAGTGTCT TTATCATTTGACTATCAATT AATCTTTATT TGGCAAGGCT 3720 TGATATATTT GAGTTACTCT TAGGTATTTGCAAGCAACTG ATCTTTCTTT TATCCCGTTT 3780 CTCCGTTAAA CCTCATTAGA AATATATTATAATGTCACCT ACTCTGTGGT TTAAGACATT 3840 CCCTTACATT ATAAGGTATT TCACGTCGTATCAGGTCGAA AAAAATAATG GTACGCTCTT 3900 TCTTATCACA AATTTCTCTC AACTTCTAGACCAATTGAAT CTTGTCTCCA ATAAGCATTG 3960 CTTTTACTGT ATGTTTCTCT CTATCAATTCAAGGCTCAAG CCATCAAATC GCTTGGTATT 4020 TCTCGCTCCC ATTTAACCAA TCGAGCTGTTAGCTCTGCTA AAGTCTCATT CTTCAGCTTC 4080 TCAAACCACT CAGAGCTTCT CCAACCAAAAATGTAGAAAA AATCTTTGAT CCACATGTAA 4140 AACCCCAGAA TTGTCATTCG AGTGAAGGAAGGTGCTGGAA ACGCGACATC AAACACGGAT 4200 TTGCCAGCTG ATTGTCATAG GAAAAAGAAAAGTTGATCAG ATAGTTCGTC CGGGGTATTT 4260 CCTACCTCGC CTTTGATCAT AGCTCTAGGCTTGCGAAACT AAGTTATGCA CGAGGCCCCC 4320 TGCGAATCCA TAAATCTAAG AAGCATGCCACAACAAACGG GATAACTTCC AGCTAAGAGA 4380 TCTATTTTGA TCTTACCCAC AAACTAGCTCCGTGTGAATT GTCCATTTCA TCAACTCCAA 4440 AGGCAGGAAA GAAGACTCTA TTCAGAGAGCAGCAAAAGAG CTC 4483 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4699 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAGCTCCCTT GGGGGGCAAG GGCAAAACTTTTTGCTAAAT GGAAAAATAT TATACCAAGT 60 GTTTGTAATA GTTACTCAAT TTGAATTAACAAAGGGGCAA ATTTGACTAT TTTGCCCTTA 120 TATCTTTTGG TCACAAAAAC ATAAAATATCCCATCCGAAA TTCCAAATGG TCCATTATCG 180 GCAAGTAGCT TTCTTTAATT ATAGTTAGTTGACAAAACAC TATCAAGATA TCATTATTAT 240 AATAATAACT TCAAAGTCCA TCATCTTAGCTGCCTCCTCA GTAGAGCCGC CAGTAAAATA 300 AGACCGATCA AATAAAAGCC GCCATTAAAATAATGAATTT TAGGACTCTC GATTGGCACG 360 TAAGTGCCAA AACTCTTCCA ATACTTTGCTGCAACTTGGG GCTGCTAGGT TCTGAGCTTC 420 CAGATATGGG ATATTTCTAA GTTTATCTCCTAATTTACAT CTCAACTAAT ATTAAGAAAT 480 TAAACAGGTA CAGCAAATCA TAAAATTTTCCTCTAAAGAA GACAATGAAT CCGGTTACTG 540 ATTCATTGGC CTTTTCAGAG TCTGCATGCCATATTCACTA AGGGGTCGTT TGGTACAAGA 600 AATAATAATA ATAATTTCGG GATAGAATTTGAGATTGCAT TTATCTTGTG TTTAATTATA 660 AGTATTAGCT AATTTCAGAA TAAATTTTACACTAAAATAG TAAAATCAAC TATCACATGT 720 AGAAGGTGGA ATGGAATAGC TAATCCCATAGCCACTCACA TAGAATATCC TTATTTATCT 780 CACTATTTTA CCAAATGATC GGTTAGTCTTCATGAGAATC CAGTATCCTC AATAAATGCA 840 GTAAGAAGTT AGAAAATTTT CATTAAATCAATTCATATAA TTTAAAAATA TTAGATATGG 900 AGCACTTAAG ATACAATAAA AGATGTACCGTTAATAATAA AAGATAAGAT AGAGTTTTAA 960 ATAGGAAAAA AAAAACGGTT CGAGACACTCTTATGGAAGG CGTTGTCTTC AAAGTAGATT 1020 CTCATTCATT GCTCTGGTGC AATAGCAAAATGACATCTTA CTCTTAAGAT ACAGCGAGCC 1080 ACTCTACAAT CTTCTATTGT ATACTCAAATGAAAGTTTTA GAGAACTTTC AAATCTCTCA 1140 ACTACTTTTA AGGGAATTCA AAATACGACCAATATTTATT ACTTACTTAC TTATAGTTAA 1200 ATGATATGAA TTTTATTTTA AATTTGAATTGAAAATATTA AATTACTTGA TTTAATATAA 1260 ACAATAGATA TCGCTAAGTA TTTACCACAAACATGGAGAT ACTACAGAAG ATTTTATTAT 1320 TTGTAACGAT GATTAAGCAG CTATTCATCTGGTTGTGCAG GATGAAAGAA AGTAACTAGC 1380 TATAATTTCT TTTGTAAAGT CAAGATAGTACGGCACCTTT GGANNAAATA TAAAACTTGT 1440 ACATCATCAA ACTCATTTTC TTTTTTCCACAAAATGATCA AGTTGACATG TTAATAGCCA 1500 GGTCAGCCGG GGCGGCTCTT AACTTCATTAGCCTACGAAT AACAAATACT CCAATAATAT 1560 TCCACTCGAA TATTTACATT TACACAAGGCTATCTCAATA TAAATAGCTC ATTGTTCATC 1620 TTAATTCTTC CAACAAGTCT TCTCATCATGTCTACCTCAG ATAAACATAA TACTCCTCAA 1680 ATGGCTGCTA TCACACTGCT AGGATTACTACTTGTTGCCA GCACCATTGA GATAGCAGGT 1740 TTCTGGTCAA ATATTTGAAC TTCCGAGCCAAAAATATTGT CTTATAATTT TGTTGGTGCA 1800 AAATCTTAAT TTAGTTGATA GTTATTTGCTTATTTTTCTT ATCAAATTGC TTGTGTTTTT 1860 TTCTCAAATT TACTTGCACT ATATTCATTTAGCGATAGTT ATTTGCTCTT TTTTCGGGTA 1920 ACACTCACTC ACAAGCTTTT CAAATTTGAGGGGAGGGCGG TGAATCTAAA ATTTGAAATT 1980 TATGAGTTTA GACTAGTGTC CATTTGATTTATTGGCTAGA CGTCTATTAG TTGTATAGTA 2040 AATCTTTTAA CACATACACC GACCTGAGTCAAAGCTATTA GGTTCGTATT AACACATAAC 2100 ACATATTCCC TCTGTTCTAA TTTATGTGACACACTTTCTG TTAGTTTCTT CCAAAGAGAA 2160 TGACATATTT GATATTTAAA AATATTTTAACTTTAAACTT TTTATTCTCA ACCTTTTATA 2220 ACATCACAAA TATTATGGAA CATATTAGACTACAAGTTCC AAATATCTTA TAGTCTGTAC 2280 AAATATTATA GCATGTTTAA TAACACAATTTTCACATTCT TCTTTTTCTT AAACTTTGTG 2340 CCGAATTAAA TTATGTCACA TAAATTAAAATGGTTACATC ATCCCCTAGT GGAGGGACCT 2400 ACCATGTCTA CTGTAAGTTT TTAACTTTTCAAGAATTACA TAATTGATTT AGTTTCTAAC 2460 ACTAATTCTA ATTCTTATTG AGCAGGGGCTCAATCAATAG GTGTTTGCTA TGGAATGCTA 2520 GGCAACAACT TGCCAAATCA TTGGGAAGTTATACAGCTCT ACAAGTCAAG AAACATAGGA 2580 AGACTGAGGC TTTATGATCC AAATCATGGAGCTTTACAAG CATTAAAAGG CTCAAACATT 2640 GAAGTTATGT TAGGACTTCC CAATTCAGATGTCAAGCACA TTGCTTCCGG AATGGAACAT 2700 GCAAGATGGT GGGTACAAAA AAATGTTAAAGATTTCTGGC CAGATGTTAA GATTAAGTAT 2760 ATTGCTGTTG GGAATGAAAT CAGCCCTGTCACAGGCACAT CTTACCTTAC CTCATTTCTT 2820 ACTCCTGCCA TGGTAAATAT TTACAAAGCAATTGGTGAAG CTGGTTTAGG AAACAACATC 2880 AAGGTCTCAA CTTCTGTAGA CATGACCTTGATTGGAAACT CTTATCCACC ATCACAGGGT 2940 TCGTTTAGGA ACGATGCTAG GTGGTTTACTGATCCAATTG TTGGGTTCTT AAGGGACACA 3000 CGTGCACCTT TACTCGTTAA CATTTACCCCTATTTCAGCT ATTCTGGTAA TCCAGGGCAG 3060 ATTTCTCTCC CCTATTCTCT TTTTACAGCACCAAATGTGG TAGTACAAGA TGGTTCACGC 3120 CAATATAGGA ACTTATTTGA TGCAATGCTGGATTCTGTGT ATGCTGCCCT CGAGCGATCA 3180 GGAGGGGCAT CTGTAGGGAT TGTTGTGTCCGAGAGTGGCT GGCCATCTGC TGGTGCATTT 3240 GGAGCTACAT ATGACAATGC AGCAACTTACTTGAGGAACT TAATTCAACA CGCTAAAGAG 3300 GGTAGCCCAA GAAAGCCTGG ACCTATTGAGACCTATATAT TTGCCATGTT TGATGAGAAC 3360 AACAAGAACC CTGAACTGGA GAAACATTTTGGATTGTTTT CCCCCAACAA GCAGCCCAAA 3420 TATAATCTCA ACTTTGGGGT CTCTGGTGGTGTTTGGGACA GTTCAGTTGA AACTAATGCT 3480 ACTGCTTCTC TCATAAGTGA GATGTGAGATGAGACACTTG AAATCTCTTT ACATAAGTAT 3540 TGCTTAGATG GAAAGCTTAG TAAAAACAAGAGAAATTTAT TCTTCATGCA AGACACTAAA 3600 TAACATTGCA CGTCTCTGTA AGTCCTCATGGATTGTTATC CAGAGACGAT GCAACTCTGA 3660 GTGGTTTTAA ATCCCTTTTC TTTGTGATGTTGGTAATTTG GCAAGAAACT TTCTGTAAGT 3720 TTGTGAATTT CATGACTTTC TGTAAGTTTGTGAATTTCAT GCACCATCAA TTATACATCT 3780 TTTCCATGTT TGATCACATT AGGATTTGGTAATTGCAAAG TTTAGTTATT TATTAATTAG 3840 TGTCTTTATC ATTTGACTCG ATCAATTAATCTTTAATTGG TAAGGCTTGA TATATCGGAG 3900 CGACTCTTAG GTAGTGGCAT TCAACTGATCTTTCTGTTAT CCCATGTCTC CGTTAACCCT 3960 CATTAGAAAT ATATTATAAT GTCACCTAAATCAGAGGTTT TAGAGCTTCA AAATCGATTG 4020 ACAACAGTTT TGGAGTTACA CTGTGGTTTAGGACATTCTG TTACATTATA AGGTATTTCA 4080 CGTCGTATCA AGGTCGAATA AAATAATGGTACGCTCTTTC TTATCACAAA TTTCTCTCAA 4140 CTTCTAGACC AATTGAATCT TGTCTCCAATAAGTATTGCT TTTACTCTAT GTTTCTCTCT 4200 ATCAATTCAA GGCTCAAGCC ATCAAATCGCTTGGTATTTC TCGCTCTCAA TTAACCAATC 4260 GAGCTGTTAA CTCTGCTAAA GTCTCATTCTTCAGCTTCTC AAACCACTCA AAGCTTCTCC 4320 AACAAAAAAA GTAGAAAAAA ACTTTGATCCACATGTAAAA CCCCATAACC ATGTCATTCG 4380 AGTGAAGGAA GGTGCTTGAA ATGCAACGAACAAACACGGC TTTGCCACTG CTTGTGATAG 4440 GTAAAAGAAA ACTTGATTAG ATAGTTCGTGCGGGGCATTT CCTACCTCGC CTTTGATCTT 4500 AGCTTTAGGC TTGCGAAACT AAGTAGAACCTAAGGCCCCA GCGAATCCGT AAATCTGAGG 4560 AGCATGCCAC AGCAAAAAGG TTAGCTTTCAGATAAGAGAT CTATTTGATC TTACCCACTA 4620 ACTAGCTCTG TGTGAATTGT CCATTTCATCAACTCCAAAG GCAGGAAAGA AGACTATTGA 4680 GAGAGCAGCA AAAGAGCTC 4699 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1020 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 7: AAAAAAAAAA AAAAACATAA GAAAGTACAG AGGAAAATGG AGTTTTCTGGATCACCAATG 60 GCATTGTTTT GTTGTGTGTT TTTCCTGTTC TTAACAGGGA GCTTGGCACAAGGCATTGGT 120 TCTATTGTAA CGAGTGACTT GTTCAACGAG ATGCTGAAGA ATAGGAACGACGGTAGATGT 180 CCTGCCAATG GCTTCTACAC TTATGATGCA TTCATAGCTG CTGCCAATTCCTTTCCTGGT 240 TTTGGAACTA CTGGTGATGA TACTGCCCGT AGGAAAGAAA TTGCTGCCTTTTTCGGTCAA 300 ACTTCTCACG AAACTACTGG TGGATCCCTG AGTGCAGAAC CATTTACAGGAGGGTATTGC 360 TTTGTTAGGC AAAATGACCA GAGTGACAGA TATTATGGTA GAGGACCCATCCAATTGACA 420 AACCGAAATA ACTATGAGAA AGCTGGAACT GCAATTGGAC AAGAGCTAGTTAACAACCCT 480 GATTTAGTGG CCACAGATGC TACTATATCA TTCAAAACAG CTATATGGTTTTGGATGACA 540 CCACAGGACA ACAAGCCATC TTCCCACGAC GTTATCATCG GTCGTTGGACTCCGTCTGCC 600 GCGGATCAGG CGGCGAATCG AGTACCAGGT TACGGTGTAA TTACCAACATCATTAACGGT 660 GGAATTGAAT GTGGCATAGG ACGGAATGAC GCAGTGGAAG ATCGAATTGGATACTACAGG 720 AGGTATTGTG GTATGTTAAA TGTTGCTCCG GGGGAAAACT TGGACTGTTACAACCAAAGG 780 AACTTCGGCC AGGGCTAGGC TTCGTTACAT AGAATGCAGA TCATGTTATGTATACAAGTT 840 ATATTTGTAT TAATTAATGA ATAAGGGGAT TGTGTATCCA TTAAGAATTAGGTGAAATAT 900 TTCTGTTATT TGTCTTCTTG GGAAGAACCA ATAGCTCCTA TATATGAGGCGCTTTTAAGT 960 GATGAGGCTA CTGCATTGAT GAAAACGAAA TTTCTATCCA GAAATAAAAGTTCCTTGTCT 1020 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1107 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8: CAGGTGCTCA AGCAGGAGTT TGTTATGGAAGGCAAGGGAA TGGATTACCA TCTCCAGCAG 60 ATGTTGTGTC GCTATGCAAC CGAAACAACATTCGTAGGAT GAGAATATAT GATCCTGACC 120 AGCCAACTCT CGAAGCGCTT AGAGGCTCCAACATTGAGCT CATGCTAGGT GTCCCGAATC 180 CGGACCTTGA GAATGTTGCT GCTAGCCAAGCCAATNCAGA TACTTGGGTC CAAAACAATG 240 TTAGGAACTA TGGTAATGTC AAGTTCAGGTATATAGCAGT TGGAAATGAA GTTAGTCCCT 300 TAAATGAAAA CTCTAAGTAT GTACCTGTCCTTCTCAACGC CATGCGAAAC ATTCAAACTG 360 CCATATCTGG TGCTGGTCTT GGAAACCAGATCAAAGTCTC CACAGCTATT GAAACTGGAC 420 TTACTACAGA CACTTCTCCT CCATCAAATGGGAGATTCAA AGATGATGTT CGACAGTTTA 480 TAGAGCCTAT CATCAACTTC CTAGTGACCAATCGCGCCCC TTTGCTTGTC AACCTTTATC 540 CTTACTTTGC AATAGCAAAC AATGCAGATATTAAGCTTGA GTATGCACTT TTTACATCCT 600 CTGAAGTTGT TGTAAATGAT AACGGAAGAGGATACCGAAA CCTTTTTGAT GCCATCTTAG 660 ATGCCACATA CTCGGCCCTT GAAAAGGCTAGTGGCTCGTC TTTGGAGATT GTTGTATCAG 720 AGAGTGGTTG GCCTTCAGCT GGAGCAGGACAATTAACATC CATTGACAAT GCCAGGACTT 780 ATAACAACAA CTTGATTAGT CACGTGAAGGGAGGGAGTCC CAAAAGGCTT CCGGTCCAAT 840 AGAGACCTAC GTTTTCGCTC TGTTTGATGAAGATCAGAAA GACCCTGAAA TTGAGAAGCA 900 TTTTGGACTA TTTTCAGCAA ACATGCAACCAAAGTACCAG ATCAGTTTTA ACTAGTTAAA 960 AGCAAGAGGA GAGCATTAAT AGGAATAAGGACTTTCCTTT GTATGAAGAG AAAGTAGTCC 1020 ATTGGCACTA TGTACTGAAA CTATATATCATGCTCATAAA GAAAGCAGTT ATTACAATAA 1080 TGAAACACTT ACAAGAAAAG CCATCAA 1107(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 809 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 9: ATTCAAGATA CAACATTTCT CCTATAGTCA TGGGATTTGT TCTCTTTTCACAATTGCCTT 60 CATTTCTTCT TGTCTCTACA CTTCTCTTAT TCCTAGTAAT ATCCCACTCTTGCCGTGCCC 120 AAAATTCTCA ACAAGACTAT TTGGATGCCC ATAACACAGC TCGTGCAGATGTAGGTGTAG 180 AACCTTTGAC CTGGGACGAC CAGGTAGCAG CCTATGCGCA AAATTATGCTTCCCAATTGG 240 CTGCAGATTG TAACCTCGTA CATTCTCATG GTCAATACGG CGAAAACCTAGCTGAGGGAA 300 GTGGCGATTT CATGACGGCT GCTAAGGCTG TTGAGATGTG GGTCGATGAGAAACAGTATT 360 ATGACCATGA CTCAAATACT TGTGCACAAG GACAGGTGTG TGGACACTATACTCAGGTGG 420 TTTGGCGTAA CTCGGTTCGT GTTGGATGTG CTAGGGTTCA GTGTAACAATGGAGGATATG 480 TTGTCTCTTG CAACTATGAT CCTCCAGGTA ATTATAGAGG CGAAAGTCCATACTAATTGA 540 AACGACCTAC GTCCATTTCA CGTTAATATG TATGGATTGT TCTGCTTGATATCAAGAACT 600 TAAATAATTG CTCTAAAAAG CAACTTAAAG TCAAGTATAT AGTAATAGTACTATATTTGT 660 AATCCTCTGA AGTGGATCTA TAAAAAGACC AAGTGGTCAT AATTAAGGGGAAAAATATGA 720 GTTGATGATC AGCTTGATGT ATGATCTGAT ATTATTATGA ACACTTTTGTACTCATACGA 780 ATCATGTGTT GATGGTCTAG CTACTTGCG 809 (2) INFORMATION FORSEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 771 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:GTTCAAAATA AAACATTTCT CCTATAGTCA TGGGATTTTT TCTCTTTTCA CAAATGCCCT 60CATTTTTTCT TGTCTCTACA CTTCTCTTAT TCCTAATAAT ATCTCACTCT TCTCATGCCC 120AAAACTCTCA ACAAGACTAT TTGGATGCCC ATAACACAGC TCGTGCAGAT GTAGGCGTGG 180AACCATTAAC TTGGGACAAC GGGGTAGCAG CCTATGCACA AAATTATGTT TCTCAATTGG 240CTGCAGACTG CAACCTCGTA CATTCTCATG GCCAATACGG CGAAAACCTA GCTCAGGGAA 300GTGGCGATTT TATGACGGCT GCTAAGGCCG TCGAGATGTG GGTCGATGAG AAACAGTACT 360ATGACCATGA CTCAAATACT TGTGCACAAG GACAGGTGTG TGGACACTAT ACTCAGGTGG 420TTTGGCGTAA CTCGGTTCGT GTTGGATGTG CTAGGGTTAA GTGCAACAAT GGAGGATATG 480TTGTCTCTTG CAACTATGAT CCTCCAGGTA ATGTCATAGG CCAAAGTCCA TACTAATTGA 540AATGAATGTC CATTTCACGT TATATATGTA TGGACTTCTG CTTGATATAT ATAAACAACT 600TAAATAATTG CACTAAAAAG CAACTTATAG TTAAAAGTAT ATAATATTTG TAATCCTCTG 660AAGAACTGGA TCTGTAAAAA GTCCAAGTGG TCTTAATTAA GGGGGGGAGG ATATATGAAT 720TCAGCTTGAT GTATGATCTG ATATTATTAT GAACTCTTTA GTACTCTTAC G 771 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:696 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 11: GTTCAAAATA AAACATTTCT CCTATAGTCA TGGAATTTGT TCTCTTTTCACAAATGTCTT 60 CATTTTTTCT TGTCTCTACG CTTCTCTTAT TCCTAATAAT ATCCCACTCTTGTCATGCTC 120 AAAACTCTCA ACAAGACTAT TTGGATGCCC ATAACACAGC TCGTGCAGATGTAGGTGTAG 180 AACCTTTGAC CTGGGACGAC CAGGTAGCAG CCTATGCACA AAATTATGCTTCCCAATTGG 240 CTGCAGATTG TAACCTCGTA CATTCTCATG GTCAATACGG CGAAAACCTAGCTTGGGGAA 300 GTGGCGATTT CTTGACGGCC GCTAAGGCCG TCGAGATGTG GGTCAATGAGAAACAGTATT 360 ATGCCCACGA CTCAAACACT TGTGCCCAAG GACAGGTGTG TGGACACTATACTCAGGTGG 420 TTTGGCGTAA CTCGGTTCGT GTTGGATGTG CTAGGGTTCA GTGTAACAATGGAGGATATA 480 TTGTCTCTTG CAACTATGAT CCTCCAGGTA ATGTTATAGG CAAAAGCCCATACTAATTGA 540 AAACATATGT CCATTTCACG TTATATATGT GTGGACTTCT GCTTGATATATATCAAGAAC 600 TTAAATAATT GCGCTAAAAA GCAACTTATA GTTAAGTATA TAGTACTATATTTGTAATTC 660 TCTGAAGTGG ATATATAATA AGACCTAGTG CTCTTG 696 (2)INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:968 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 12: GAAAATGGAG TTTTCTGGAT CACCACTGAC ATTGTTTTGT TGTGTGTTTTTCCTGTTCCT 60 AACAGGGAGC TTGGCACAAG GCATTGGCTC AATTGTAACG AATGACTTGTTCAACGAGAT 120 GCTGAAGAAT AGGAACGACG GTAGATGTCC TGCCAATGGC TTCTATACTTATGATGCATT 180 CATAGCTGCT GCCAATTCCT TTCCTGGTTT TGGAACTAGT GGTGATGATACTGCCCGTAG 240 GAAAGAAATT GCTGCCTTTT TCGGTCAAAC TTCTCATGAA ACTACAGGTGGTTCCCTGAG 300 TGCAGAACCT TTTACAGGAG GATATTGCTT TGTTAGGCAA AATGACCAGAGTGACAGATA 360 TTATGGTAGA GGACCCATCC AATTGACAAA CCAAAATAAC TATGAGAAAGCTGGAAATGC 420 AATTAGACAA GACCTAGTTA ACAACCCAGA TTTAGTAGCT ACAGATGCTACTATATCATT 480 CAAAACAGCT ATATGGTTCT GGATGACACC ACAGGATAAT AAGCCATCAAGCCACGACGT 540 TATCATCGGT AGTTGGACTC CGTCCGCCGC TGATCAGTCG GCGAATCGAGCACCTGGTTG 600 CGGTGTAATT ACCAACATTA TTAACGGTGG AATTGAATGT GGCGTAGGTCCGAATGCCGC 660 AGTGGAAGAT CGAATTGGAT ACTACAGGAG GTATTGTGGT ATGTTGAATGTTGCTCCTGG 720 GGACAACTTG GACTGTTACA ACCAAAGGAA CTTCGCCCAA GGCTAGGATTCGTTAGATCA 780 TGTTATGTGT ACACAAGTTA TATTTGTATG TAATGAATAA GGGGATTGTGTACCCATTTA 840 GAATAAGGGG AAATATTTCT GTTATTTGTC TTCTTCGAAA GAATAACCAGTAGTTCCTAT 900 ATATCTGGTG CTTCGAGTGA AAACGAATAT TCTATCCGGA AATAAATACTGTATGTTTCT 960 TGTCTTAT 968 (2) INFORMATION FOR SEQ ID NO: 13: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1108 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CAGGTGCTCA AGCAGGAGTTTGTTATGGAA GGCAAGGGAA TGGATTACCA TCTCCAGCAG 60 ATGTTGTGTC GCTATGCAACCGAAACAACA TTCGTAGGAT GAGAATATAT GATCCTGACC 120 AGCCAACTCT CGAAGCGCTTAGAGGCTCCA ACATTGAGCT CATGCTAGGT GTCCCGAATC 180 CGGACCTTGA GAATGTTGCTGCTAGCCAAG CCAATGCAGA TACTTGGGTC CAAAACAATG 240 TTAGGAACTA TGGTAATGTCAAGTTCAGGT ATATAGCAGT TGGAAATGAA GTTAGTCCCT 300 TAAATGAAAA CTCTAAGTATGTACCTGTCC TTCTCAACGC CATGCGAAAC ATTCAAACTG 360 CCATATCTGG TGCTGGTCTTGGAAACCAGA TCAAAGTCTC CACAGCTATT GAAACTGGAC 420 TTACTACAGA CACTTCTCCTCCATCAAATG GGAGATTCAA AGATGATGTT CGACAGTTTA 480 TAGAGCCTAT CATCAACTTCCTAGTGACCA ATCGCGCCCC TTTGCTTGTC AACCTTTATC 540 CTTACTTTGC AATAGCAAACAATGCAGATA TTAAGCTTGA GTATGCACTT TTTACATCCT 600 CTGAAGTTGT TGTAAATGATAACGGAAGAG GATACCGAAA CCTTTTTGAT GCCATCTTAG 660 ATGCCACATA CTCGGCCCTTGAAAAGGCTA GTGGCTCGTC TTTGGAGATT GTTGTATCAG 720 AGAGTGGTTG GCCTTCAGCTGGAGCAGGAC AATTAACATC CATTGACAAT GCCAGGACTT 780 ATAACAACAA CTTGATTAGTCACGTGAAGG GAGGGAGTCC CAAAAGGCCT TCCGGTCCAA 840 TAGAGACCTA CGTTTTCGCTCTGTTTGATG AAGATCAGAA AGACCCTGAA ATTGAGAAGC 900 ATTTTGGACT ATTTTCAGCAAACATGCAAC CAAAGTACCA GATCAGTTTT AACTAGTTAA 960 AAGCAAGAGG AGAGCATTAATAGGAATAAG GACTTTCCTT TGTATGAAGA GAAAGTAGTC 1020 CATTGGCACT ATGTACTGAAACTATATATC ATGCTCATAA AGAAAGCAGT TATTACAATA 1080 ATGAAACACT TACAAGAAAAGCCATCAA 1108 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1195 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 14: CTCAATTCTT GTTTTCCTTA CAAATGGCACATTTAATTGT CACACTGCTT CTCCTTAGTG 60 TACTTACATT AGCTACCCTG GATTTTACAGGTGCTCAAGC AGGAGTTTGT TATGGAAGGC 120 AAGGGAATGG ATTACCATCT CCAGCAGATGTTGTGTCGCT ATGCAACCGA AACAACATTC 180 GTAGGATGAG AATATATGAT CCTGACCAGCCAACTCTCGA AGCGCTTAGA GGCTCCAACA 240 TTGAGCTCAT GCTAGGTGTC CCGAATCCGGACCTTGAGAA TGTTGCTGCT AGCCAAGCCA 300 ATGCAGATAC TTGGGTCCAA AACAATGTTAGGAACTATGG TAATGTCAAG TTCAGGTATA 360 TAGCAGTTGG AAATGAAGTT AGTCCCTTAAATGAAAACTC TAAGTATGTA CCTGTCCTTC 420 TCAACGCCAT GCGAAACATT CAAACTGCCATATCTGGTGC TGGTCTTGGA AACCAGATCA 480 AAGTCTCCAC AGCTATTGAA ACTGGACTTACTACAGACAC TTCTCCTCCA TCAAATGGGA 540 GATTCAAAGA TGATGTTCGA CAGTTTATAGAGCCTATCAT CAACTTCCTA GTGACCAATC 600 GCGCCCCTTT GCTTGTCAAC CTTTATCCTTACTTTGCAAT AGCAAACAAT GCAGATATTA 660 AGCTTGAGTA TGCACTTTTT ACATCCTCTGAAGTTGTTGT AAATGATAAC GGAAGAGGAT 720 ACCGAAACCT TTTTGATGCC ATCTTAGATGCCACATACTC GGCCCTTGAA AAGGCTAGTG 780 GCTCGTCTTT GGAGATTGTT GTATCAGAGAGTGGTTGGCC TTCAGCTGGA GCAGGACAAT 840 TAACATCCAT TGACAATGCC AGGACTTATAACAACAACTT GATTAGTCAC GTGAAGGGAG 900 GGAGTCCCAA AAGGCCTTCC GGTCCAATAGAGACCTACGT TTTCGCTCTG TTTGATGAAG 960 ATCAGAAAGA CCCTGAAATT GAGAAGCATTTTGGACTATT TTCAGCAAAC ATGCAACCAA 1020 AGTACCAGAT CAGTTTTAAC TAGTTAAAAGCAAGAGGAGA GCATTAATAG GAATAAGGAC 1080 TTTCCTTTGT ATGAAGAGAA AGTAGTCCATTGGCACTATG TACTGAAACT ATATATCATG 1140 CTCATAAAGA AAGCAGTTAT TACAATAATGAAACACTTAC AAGAA AA: (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 584 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (A)NAME/KEY: CDS (B) LOCATION: 37..321 (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 15: ACATCAAAAA CTTGAAACTC CAAATAGCTC ATCAAA ATG TTT TCC AAA ACT AAT54 Met Phe Ser Lys Thr Asn 1 5 CTT TTT CTT TGC CTT TCT TTG GCT ATT TTGGTA ATA GTA ATA TCC TCA 102 Leu Phe Leu Cys Leu Ser Leu Ala Ile Leu ValIle Val Ile Ser Ser 10 15 20 CAA GTT GAT GCA AGG GAG ATG TCT AAG GCG CCTGCT TCA ATA ACC CAA 150 Gln Val Asp Ala Arg Glu Met Ser Lys Ala Pro AlaSer Ile Thr Gln 25 30 35 GCA ATG AAT TCA AAC ATC ATT ACT GAT CAG AAG ATGGGT GCA GGA ATC 198 Ala Met Asn Ser Asn Ile Ile Thr Asp Gln Lys Met GlyAla Gly Ile 40 45 50 ACC CGT AAG ATA CCG GGT TGG ATA CGA AAA GGT GCA AAACCT GGA GGC 246 Thr Arg Lys Ile Pro Gly Trp Ile Arg Lys Gly Ala Lys ProGly Gly 55 60 65 70 AAA ATC ATT GGC AAA GCT TGC AAA ATT TGC TCA TGT AAATAC CAG ATT 294 Lys Ile Ile Gly Lys Ala Cys Lys Ile Cys Ser Cys Lys TyrGln Ile 75 80 85 TGC AGC AAA TGT CCT AAA TGT CAT GAC TAATGTACTTGTGCTGGTGT 341 Cys Ser Lys Cys Pro Lys Cys His Asp 90 95 GAGTCTAGTTTTGAGGATAA AGGGAAAGCT ATGAATAGCC TAATATAATT CTATTCACTT 401 TCCTCTAGTTAATTTCTCTT AGTTTGTGTT TTGTTTTGTT AATAGTTATT ATATTGTTGG 461 AACTTGCAACAAGTCTTGGG TCAATATATA TTCTTGTTTT CTAGTCTTTA TATTGTATGG 521 TATTGTATTGTATTGTATTT TTCTTTAGTC ACGTGATATT TGAAACCAAA TCTGATTAAA 581 TCT 584 (2)INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:502 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:CDS (B) LOCATION: 39..320 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:AGCAAAAAAT TAAAACTCGA TATAGCTCAT CTTTCAAA ATG TTT TCC AAA ACT 53 Met PheSer Lys Thr 1 5 ATT CTT TTT CTT TGC TTT TCT TTG GCT ATT TTG GTA ATG GTAATA TCC 101 Ile Leu Phe Leu Cys Phe Ser Leu Ala Ile Leu Val Met Val IleSer 10 15 20 TCA CAA GCT GAT GCA AGG GAG ATG TCT AAG GCG GCT GCT CCA ATTACC 149 Ser Gln Ala Asp Ala Arg Glu Met Ser Lys Ala Ala Ala Pro Ile Thr25 30 35 CAA GCA ATG AAT TCA AAC ATC ATT ACT GAT CAG AAG ACG GGT GCA GGA197 Gln Ala Met Asn Ser Asn Ile Ile Thr Asp Gln Lys Thr Gly Ala Gly 4045 50 ATC ATC CGT AAG ATA CCG GGT TGG ATA CGA AAA GGT GCA AAA GGA GGC245 Ile Ile Arg Lys Ile Pro Gly Trp Ile Arg Lys Gly Ala Lys Gly Gly 5560 65 AAC ATC ATT GGC AAA GCT TGC AAA ATT TGC TCA TGT AAA TAC CAG ATT293 Asn Ile Ile Gly Lys Ala Cys Lys Ile Cys Ser Cys Lys Tyr Gln Ile 7075 80 85 TGC AGC AAA TGT CCT AAA TGT CAT GAC TAATGTACTT GTGTTGGTGT 340Cys Ser Lys Cys Pro Lys Cys His Asp 90 GAGTCTAGTT TTGAGAATAA AGGGAAAGCTATGAATAGCC TAATATAATT CTATCACTTT 400 CCTCTAGTTA ATTTCTCTTA GTTTGTGTTTTGTTAATAGT TATTATATTG TTGGAACTTG 460 CAACAAGTCT TGGGTCAATA TATATCTTGTTTTCTAGTCT TT 502 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 560 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 38..322 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 17: GACAGTAAAA AACTGAAACT CCAAATAGCT CATCAAA ATGTTT TCC AAA ACT AAC 55 Met Phe Ser Lys Thr Asn 1 5 CTT TTT CTT TGC CTTTCT TTG GCT ATT TTG CTA ATT GTA ATA TCC TCA 103 Leu Phe Leu Cys Leu SerLeu Ala Ile Leu Leu Ile Val Ile Ser Ser 10 15 20 CAA GCT GAT GCA AGG CAGATT TCT AAG GCG GCT GCT CCA ATT ACC CAT 151 Gln Ala Asp Ala Arg Gln IleSer Lys Ala Ala Ala Pro Ile Thr His 25 30 35 GCA ATG AAT TCA AAC AAC ATTACT AAT CAG AAG ACG GGT GCC GGA ATC 199 Ala Met Asn Ser Asn Asn Ile ThrAsn Gln Lys Thr Gly Ala Gly Ile 40 45 50 ATC CGT AAG ATA CCG GGT TGG ATACGA AAA GGT GCA AAA CCA GGA GGC 247 Ile Arg Lys Ile Pro Gly Trp Ile ArgLys Gly Ala Lys Pro Gly Gly 55 60 65 70 AAA GTC GCC GGC AAA GCT TGT AAAATT TGC TCA TGT AAA TAC CAG ATT 295 Lys Val Ala Gly Lys Ala Cys Lys IleCys Ser Cys Lys Tyr Gln Ile 75 80 85 TGC AGC AAA TGT CCT AAA TGT CAT GACTAAAGTTAGG CCTTGAGACT 342 Cys Ser Lys Cys Pro Lys Cys His Asp 90 95ATGTACTTGT GCTGGTGTGA GTTTAATTTT GAGAGTAAAG GGAAAGTTAT GAATAGCCTA 402ATATAATTCT ATTCACTATG TTTTCTTAGT AATTCTTATT GTTGAAACTT GGAACAGGTC 462TTTGGGTCAA AATGTACCTC TTGTCTTGTA GTCTTTCAAC TGTATGGTAT TGTACTGTAT 522CTTTCTTTAG CCACTTGATA TCAAATCCGA TTAAATCT 560 (2) INFORMATION FOR SEQ IDNO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 529 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:37..321 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ACAGTAAAAA ACTGAAACTCCAAATAGCTC ATCAAA ATG TTT TCC AAA ACT AAC 54 Met Phe Ser Lys Thr Asn 5CTT TTT CTT TGC CTT TCT TTG GCT ATT TTG CTA ATT GTA ATA TCC TCA 102 LeuPhe Leu Cys Leu Ser Leu Ala Ile Leu Leu Ile Val Ile Ser Ser 10 15 20 CAAGCT GAT GCA AGG GAG ATG TCT AAG GCG GCT GTT CCA ATT ACC CAA 150 Gln AlaAsp Ala Arg Glu Met Ser Lys Ala Ala Val Pro Ile Thr Gln 25 30 35 GCA ATGAAT TCA AAC AAC ATT ACT AAT CAG AAG ACG GGT GCC GGA ATC 198 Ala Met AsnSer Asn Asn Ile Thr Asn Gln Lys Thr Gly Ala Gly Ile 40 45 50 ATC CGT AAGATA CCG GGT TGG ATA CGA AAA GGT GCA AAA CCA GGA GGC 246 Ile Arg Lys IlePro Gly Trp Ile Arg Lys Gly Ala Lys Pro Gly Gly 55 60 65 70 AAA GTC GCCGGC AAA GCT TGT AAA ATT TGC TCA TGT AAA TAC CAG ATT 294 Lys Val Ala GlyLys Ala Cys Lys Ile Cys Ser Cys Lys Tyr Gln Ile 75 80 85 TGC AGC AAA TGTCCT AAA TGT CAT GAC TAAAGTTAGG CCTTGAGACT 341 Cys Ser Lys Cys Pro LysCys His Asp 90 95 ATGTACTTGT GCTGGTGTGA GTTTAGTTTT GAGAGTAAAG GGAAAGTTATGAATAGCCTA 401 ATATAATTGT ATTCACTATG TTTTCTTAGT AATTCTTATT GTTGAAACTTGGAACAGGTC 461 TTTGGGTCAA AATGTACCTC TTGTCTTGTA GTCTTTCAAC TGTATAGTATTGTACTGTAT 521 TTTTCTTT 529 (2) INFORMATION FOR SEQ ID NO: 19: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 607 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 32..364 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: AAAAAATTGA AACTCCAAAT AGCTCATCAA A ATG TTTTCC AAA ACT AAC CTT 52 Met Phe Ser Lys Thr Asn Leu 1 5 TTT CTT TGC CTTTCT TTG GCT ATT TTG CTA ATT GTA ATA TCC TCA CAA 100 Phe Leu Cys Leu SerLeu Ala Ile Leu Leu Ile Val Ile Ser Ser Gln 10 15 20 GCT GAT GCA AGG GAGACG TCT AAG GCA ACT GCT CCA ATT ACC CAA GAA 148 Ala Asp Ala Arg Glu ThrSer Lys Ala Thr Ala Pro Ile Thr Gln Glu 25 30 35 ATG AAT TCA AAC AAC ACTACT GAT CAG AAG ATA CCA AAA CGT CCA AAA 196 Met Asn Ser Asn Asn Thr ThrAsp Gln Lys Ile Pro Lys Arg Pro Lys 40 45 50 55 CCA GGA GGC AAT ATC TTCGGC AAA GCT TGT AAA ATT TGC CCA TGT AAA 244 Pro Gly Gly Asn Ile Phe GlyLys Ala Cys Lys Ile Cys Pro Cys Lys 60 65 70 TAC CAG ATT TGC AGC AAA TGTCCT AAA TGT GAT GAC CAA AAT ATC GCC 292 Tyr Gln Ile Cys Ser Lys Cys ProLys Cys Asp Asp Gln Asn Ile Ala 75 80 85 GGC AAA TTT TGT AAA ATT TGC TCATGT AAG ACT CAG ATT TGC AGT AAA 340 Gly Lys Phe Cys Lys Ile Cys Ser CysLys Thr Gln Ile Cys Ser Lys 90 95 100 TGT CCT AAA TGT CAT AAC CAA AATTAGGCCTCAG AGACTATGTA CTTGTGCTGG 394 Cys Pro Lys Cys His Asn Gln Asn 105110 TGTGAGTTTA GTTTTGAGAA TAAAAGGAAA GTTATGAATA GCCTAATATA ATTCTATTCA454 CTTTCCTCTA GTTAATTTCT CTTAGTTTGT GTTTTGTTTT GTTAGTAGTT CCTATTGTTG514 CAACTTGCAA CAAGTCTTGG GGTCAACATG TACCTCTTGT CTTGTAGTCT TTCGACTGTA574 TGATATTGTA CCGTATTGTA TTGTATTTTC TTT 607 (2) INFORMATION FOR SEQ IDNO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1358 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:7..1086 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: CCTCAA ATG GCT GCT ATCACA CTC CTA GGA TTA CTA CTT GTT GCC AGC 48 Met Ala Ala Ile Thr Leu LeuGly Leu Leu Leu Val Ala Ser 1 5 10 AGC ATT GAC ATA GCA GGG GCT CAA TCGATA GGT GTT TGC TAT GGA ATG 96 Ser Ile Asp Ile Ala Gly Ala Gln Ser IleGly Val Cys Tyr Gly Met 15 20 25 30 CTA GGC AAC AAC TTG CCA AAT CAT TGGGAA GTT ATA CAG CTC TAC AAG 144 Leu Gly Asn Asn Leu Pro Asn His Trp GluVal Ile Gln Leu Tyr Lys 35 40 45 TCA AGA AAC ATA GGA AGA CTG AGG CTT TATGAT CCA AAT CAT GGA GCT 192 Ser Arg Asn Ile Gly Arg Leu Arg Leu Tyr AspPro Asn His Gly Ala 50 55 60 TTA CAA GCA TTA AAA GGC TCA AAT ATT GAA GTTATG TTA GGA CTT CCC 240 Leu Gln Ala Leu Lys Gly Ser Asn Ile Glu Val MetLeu Gly Leu Pro 65 70 75 AAT TCA GAT GTG AAG CAC ATT GCT TCC GGA ATG GAACAT GCA AGA TGG 288 Asn Ser Asp Val Lys His Ile Ala Ser Gly Met Glu HisAla Arg Trp 80 85 90 TGG GTA CAG AAA AAT GTT AAA GAT TTC TGG CCA GAT GTTAAG ATT AAG 336 Trp Val Gln Lys Asn Val Lys Asp Phe Trp Pro Asp Val LysIle Lys 95 100 105 110 TAT ATT GCT GTT GGG AAT GAA ATC AGC CCT GTC ACTGGC ACA TCT TAC 384 Tyr Ile Ala Val Gly Asn Glu Ile Ser Pro Val Thr GlyThr Ser Tyr 115 120 125 CTA ACC TCA TTT CTT ACT CCT GCT ATG GTA AAT ATTTAC AAA GCA ATT 432 Leu Thr Ser Phe Leu Thr Pro Ala Met Val Asn Ile TyrLys Ala Ile 130 135 140 GGT GAA GCT GGT TTG GGA AAC AAC ATC AAG GTC TCAACT TCT GTA GAC 480 Gly Glu Ala Gly Leu Gly Asn Asn Ile Lys Val Ser ThrSer Val Asp 145 150 155 ATG ACC TTG ATT GGA AAC TCT TAT CCA CCA TCA CAGGGT TCG TTT AGG 528 Met Thr Leu Ile Gly Asn Ser Tyr Pro Pro Ser Gln GlySer Phe Arg 160 165 170 AAC GAT GCT AGG TGG TTT GTT GAT CCC ATT GTT GGCTTC TTA AGG GAC 576 Asn Asp Ala Arg Trp Phe Val Asp Pro Ile Val Gly PheLeu Arg Asp 175 180 185 190 ACA CGT GCA CCT TTA CTC GTT AAC ATT TAC CCCTAT TTC AGT TAT TCT 624 Thr Arg Ala Pro Leu Leu Val Asn Ile Tyr Pro TyrPhe Ser Tyr Ser 195 200 205 GGT AAT CCA GGC CAG ATT TCT CTC CCC TAT TCTCTT TTT ACA GCA CCA 672 Gly Asn Pro Gly Gln Ile Ser Leu Pro Tyr Ser LeuPhe Thr Ala Pro 210 215 220 AAT GTG GTG GTA CAA GAT GGT TCC CGC CAA TATAGG AAC TTA TTT GAT 720 Asn Val Val Val Gln Asp Gly Ser Arg Gln Tyr ArgAsn Leu Phe Asp 225 230 235 GCA ATG CTG GAT TCT GTG TAT GCT GCC CTC GAGCGA TCA GGA GGG GCA 768 Ala Met Leu Asp Ser Val Tyr Ala Ala Leu Glu ArgSer Gly Gly Ala 240 245 250 TCT GTA GGG ATT GTT GTG TCC GAG AGT GGC TGGCCA TCT GCT GGT GCA 816 Ser Val Gly Ile Val Val Ser Glu Ser Gly Trp ProSer Ala Gly Ala 255 260 265 270 TTT GGA GCC ACA TAT GAC AAT GCA GCA ACTTAC TTG AGG AAC TTA ATT 864 Phe Gly Ala Thr Tyr Asp Asn Ala Ala Thr TyrLeu Arg Asn Leu Ile 275 280 285 CAA CAC GCT AAA GAG GGT AGC CCA AGA AAGCCT GGA CCT ATT GAG ACC 912 Gln His Ala Lys Glu Gly Ser Pro Arg Lys ProGly Pro Ile Glu Thr 290 295 300 TAT ATA TTT GCC ATG TTT GAT GAG AAC AACAAG AAC CCT GAA CTG GAG 960 Tyr Ile Phe Ala Met Phe Asp Glu Asn Asn LysAsn Pro Glu Leu Glu 305 310 315 AAA CAT TTT GGA TTG TTT TCC CCC AAC AAGCAG CCC AAA TAT AAT ATC 1008 Lys His Phe Gly Leu Phe Ser Pro Asn Lys GlnPro Lys Tyr Asn Ile 320 325 330 AAC TTT GGG GTC TCT GGT GGA GTT TGG GACAGT TCA GTT GAA ACT AAT 1056 Asn Phe Gly Val Ser Gly Gly Val Trp Asp SerSer Val Glu Thr Asn 335 340 345 350 GCT ACT GCT TCT CTC GTA AGT GAG ATGTGAGCTGATG AGACACTTGA 1103 Ala Thr Ala Ser Leu Val Ser Glu Met 355 360AATCTCTTTA CATACGTATT CCTTGGATGG AAAACCTAGT AAAAACAAGA GAAATTTTTT 1163CTTTATGCAA GATACTAAAT AACATTGCAT GTCTCTGTAA GTCCTCATGG ATTGTTATCC 1223AGTGACGATG CAACTCTGAG TGGTTTTAAA TTCCTTTTCT TTGTGATATT GGTAATTTGG 1283CAAGAAACTT TCTGTAAGTT TGTGAATTTC ATGCATCATT AATTATACAT CAGTTCCATG 1343TTTGATCAAA AAAAA 1358 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1204 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTGTTTCTTA CTCTCTCATT TCCATTTTAGCTATGACTTT ATGCATTAAA AATGGCTTTC 60 TTGCAGCTGC CCTTGTACTT GTTGGGCTGTTAATTTGCAG TATCCAAATG ATAGGGGCAC 120 AATCTATTGG AGTATGCTAT GGAAAACATGCAAACAATTT ACCATCAGAC CAAGATGTTA 180 TAAACCTATA CGATGCTAAT GGCATCAGAAAGATGAGAAT CTACAATCCA GATACAAATG 240 TCTTCAACGC TCTCAGAGGA AGTAACATTGAGATCATTCT CGACGTCCCA CTTCAAGATC 300 TTCAATCCCT AACTGATCCT TCAAGAGCCAATGGATGGGT CCAAGATAAC ATAATAAATC 360 ATTTCCCAGA TGTTAAATTT AAATATATAGCTGTTGGAAA TGAAGTCTCT CCCGGAAATA 420 ATGGTCAATA TGCACCATTT GTTGCTCCTGCCATGCAAAA TGTATATAAT GCATTAGCAG 480 CAGCAGGGTT GCAAGATCAA ATCAAGGTCTCAACTGCAAC ATATTCAGGG ATCTTAGCGA 540 ATACCAACCC GCCCAAAGAT AGTATTTTTCGAGGAGAATT CAATAGTTTC ATTAATCCCA 600 TAATCCAATT TCTAGTACAA CATAACCTTCCACTCTTAGC CAATGTCTAT CCTTATTTTG 660 GTCACATTTT CAACACTGCT GATGTCCCACTTTCTTATGC TTTGTTCACA CAACAAGAAG 720 CAAATCCTGC AGGATATCAA AATCTTTTTGATGCCCTTTT GGATTCTATG TATTTTGCTG 780 TAGAGAAAGC TGGAGGACAA AATGTGGAGATTATTGTATC TGAAAGTGGC TGGCCTTCTG 840 AAGGAAACTC TGCAGCAACT ATTGAAAATGCTCAAACTTA CTATGAAAAT TTGATTAATC 900 ATGTGAAAAG CGGGGCAGGA ACTCCAAAGAAACCTGGAAA TGCTATAGAA ACTTATTTAT 960 TTGCCATGTT TGATGAAAAT AATAAGGAAGGAGATATCAC AGAGAAACAC TTTGGACTCT 1020 TTTCTCCTGA TCAGAGGGCA AAATATCAACTCAATTTCAA TTAATTAATG CATGGTAACA 1080 TTTATTGATA TATATAGTGA TATGAGTAATAAGGAGAAGT AGAACTGCTA TGTTTTTCTC 1140 TTCAATTGAA AATGTAACTC TGGTTTCACTTTGATATTTA TATGACATGT TTATTGAGAT 1200 CTAA 1204 (2) INFORMATION FOR SEQID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1131 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ACCAGAGAAGACCCCATTTG CAGTATCAAA AATGGGTTTA CCTAAAATGG CAGCCATTGT 60 TGTGGTGGTGGCTTTGATGC TATCACCCTC TCAAGCCCAG YTTTCTCCTT TCTTCTACGC 120 CACCACATGCCCTCAGCTGC CTTTCGTTGT TCTCAACGTG GTTGCCCAAG CCCTACAGAC 180 TGATGACCGAGCTGCTGCTA AGCTCATTCG CCTCCATTTT CATGATTGCT TTGTCAATGG 240 GTGTGATGGATCGATTCTAT TGGTAGACGT ACCGGGCGTT ATCGATAGTG AACTTAATGG 300 ACCTCCAAATGGTGGAATCC AAGGAATGGA CATTGTGGAC AACATCAAAG CAGCAGTTGA 360 GAGTGCTTGTCCAGGAGTTG TTTCTTGCGC TGATATCTTA GCCATTTCAT CTCAAATCTC 420 TGTTTTCTTGTCGGGAGGAC CAATTTGGGT TGTACCAATG GGAAGAAAAG ACAGCAGAAT 480 AGCCAATAGAACTGGAACCT CAAACTTACC TGGTCCCTCA GAAACTCTAG TGGGACTTAA 540 AGGCAAGTTTAAAGATCAAG GGCTTGATTC TACAGATCTC GTGGCTCTAT CAGGAGCCCA 600 CACGTTTGGAAAATCAAGAT GCATGTTCTT CAGTGACCGC CTCATCAACT TCAACGGCAC 660 AGGAAGACCCGACACAACGC TTGACCCAAT ATACAGGGAG CAGCTTCGAA GACTTTGTAC 720 TACTCAACAAACACGAGTAA ATTTCGACCC AGTCACACCC ACTAGATTTG ACAAGACCTA 780 TTACAACAATTTGATTAGCT TAAGAGGGCT TCTCCAAAGC GACCAAGAGC TCTTCTCAAC 840 TCCCAGAGCTGATACCACAG CCATTGTCAR AACTTTTGCT GCCAACGAAC GTGCCTTCTT 900 TAAACAATTTGTGAAATCAA TGATCAAAAT GGGCAACCTC AAGCCTCCCC CTGGCATTGC 960 ATCAGAAGTTAGATTGGACT GTAAGAGGGT CAACCCAGTC AGAGCCTACG ACGTTATGTA 1020 ATAACTTTATCCCACTTCAT CCCTTCTACT TTTGCTGTCT CTTGTACTAC TTTGTTGATG 1080 TATTAGTTCAACCGGTTAAG ATATATATAT CGTTGACCTA AATAATAGAT C 1131 (2) INFORMATION FORSEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 627 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:AATAGTTTCA TTAATCCCAT AATCCAATTT CTAGCACGAA ATAACCTTCC ACTCTTAGCC 60AATGTCTATC CTTATTTTGG TCACATTTAC AACACTGCTG ATGTCCCACT TTCTTATGCA 120TTGTTCACAC AACAAGAAGC AAATCCTGCA GGATATCAAA ATCTTTTTGA TGCCCTTTTG 180GATTCTATGT ATTTTGCTGT AGAGAAAGCT GGAGGACCAA ATGTGGAGAT TATTGTATCT 240GAAAGTGGCT GGCCTTCTGA AGGAAACTCT GCAGCAACTA TTGAAAATGC TCAAACTTAT 300TACAGAAATT TGATTGATCA TGTGAAAAGA GGGGCAGGAA CCCCAAAGAA ACCTGGAAAG 360ACTATAGAAA CTTATTTATT TGCCATGTTT GATGAAAATG ATAAGAAAGG AGAAATTACA 420GAGAAACACT TTGGACTCTT TTCTCCTGAT CAGAGGGCAA AATATCAACT CAATTTCAAT 480TAATTAATGG CAATATATAT TGATATATAT ATATAGTGAT ATGAGTAATA AGGAGAACTG 540CTATGTTTTT CTCTTCAATT GAAAATGYAA TTCTGGTTTC ACTTTGATAT CTATATGTCA 600TTTATTGAAA TCTCGTCTTT TGGTTTT 627 (2) INFORMATION FOR SEQ ID NO: 24: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 966 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AATGTCTTCA ACGCTCTCAGAGGAAGTAAC ATTGAGATCA TTCTCGACGT CCCACTTCAA 60 GATCTTCAAT CCCTAACTGATCCTTCAAGA GCCAATGGAT GGGTCCAAGA TAACATAATA 120 AATCATTTCC CAGATGTTAAATTTAAATAT ATAGCTGTTG GAAATAAAGT CTCTCCCGGA 180 AATAATGGTC AATATGCACCATTTGTTGCT CCTGCCATGC AAAATGTATA TAATGCATTA 240 GCAGCAGCAG GGTTGCAAGATCAAATCAAG GTCTCAACTG CAACATATTC AGGGATCTTA 300 GCGAATACCT ACCCGCCCAAAGATAGTATT TTTCGAGGAG AATTCAATAG TTTCATTAAT 360 CCCATAATCC AATTTCTAGTACAACATAAC CTTCCACTCT TAGCCAATGT CTATCCTTAT 420 TTTGGTCACA TTTTCAACACTGCTGATGTC CCACTTTCTT ATGCTTTGTT CACACAACAA 480 GAAGCAAATC CTGCAGGATATCAAAATCTT TTTGATGCCC TTTTGGATTC TATGTATTTT 540 GCTGTAGAGA AAGCTGGAGGACAAAATGTG GAGATTATTG TATCTGAAAG TGGCTGGCCT 600 TCTGAAGGAA ACTCTGCAGCAACTATTGAA AATGCTCAAA CTTACTATGA AAATTTGATT 660 AATCATGTGA AAAGCGGGGCAGGAACTCCA AAGAAACCTG GAAAGGCTAT AGAAACTTAT 720 TTATTTGCCA TGTTTGATGAAAATAATAAG GAAGGAGATA TCACAGAGAA ACACTTTGGA 780 CTCTTTTCTC CTGATCAGAGGGCAAAATAT CAACTCAATT TCAATTAATT AATGCATGGT 840 AACATTTATT GATATATATAGTGATATGAG TAATAAGGAG AAGTAGAACT GCTATGTTTT 900 TCTCTTCAAT TGAAAATGTAACTCTGGTTT CACTTTGATA TTTATATGAC ATATTTATTG 960 AGATCT 966 (2)INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1099 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 25: ATGGCTTTCT TGCAGCTGCC CTTGTACTTG TTGGGCTRAT AATGTGCAGTATCCAAATCA 60 TAGGGGCACA GTCTATTGGA GTATGCTATG GAAAAGCTGC CAACAATTTACCATCAGACC 120 AAGATGTTAT AAACCTATAC AATGCTAATG GCATCAGAAA GTTGAGAATTTACTATCCTG 180 ATAAAAACAT TTTCAAAGCT CTCAATGGAA GTAACATTGA GATCATTCTTGGTGTCCCAA 240 ATCAAGACCT TGAAGCCCTA GCCAATTCTT CAATAGCCAA TGGTTGGGTTCAAGATAACA 300 TAAGAAGTCA TTTCCCATAT GTTAAATTCA AGTACATATC TATAGGAAATAAAGTATCTC 360 CCACAAATAA TGATCAATAT TCAGAATTTC TTCTTCAAGC AATGAAAAATGTGTACAATG 420 CTTTAGCAGC AGCAGGGTTG CAAGATATGA TCAAGGTCTC AACTGTGACATATTCAGGGG 480 TCTTAGCGAA TACCTACCCA CCTGAACGTA GTATTTTTCG CGAAGAATTCAAGAGTTTCA 540 TTAATCCGAT AATCCAATTT CTAGCACGAA ATAACCTTCC ACTCTTAGCCAATGTCTATC 600 CTTATTTTGT TCACGTTTCC AACACTGCTG ATGTTTCACT TTCTTATGCATTGTTCACAC 660 AGCAAGGAAC AAATTCAGCA GGGTATCAAA ATCTTTTTGA TGCTATTTTGGATTCTATGT 720 ATTTTGCTGT AGAGAAAGCT GGAGGACCAA ATGTGGAGAT TATTGTATCTGAAAGTGGAT 780 GGCCTTCTGA AGGAAGCTCT GCAGCAACTA TTGAAAACGC TCAAACTTATTACAGAAATT 840 TGATTAATCA TGTGAAAAGC GGGGCAGGAA CTCCAAAGAA ACCTGGAAAGACTATAGAAA 900 CTTATTTGTT TGCCATGTTT GATGAAAATG ATAAGATAGG AGAAATCACAGAGAAACACT 960 TTGGACTGTT TTCTCCTGAT CAAAGGGCAA AATATCAACT CAATTTCAATTATTTGCCAA 1020 TATATATATT GAGATGAGTA ATAAGGACAA CTGTTATGTT TTTCTCTTCAATTGAAAATG 1080 TAACTCTGGT TTCACTTTG 1099 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1201 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GAAACTTCTCTCATTTCCAT TTTAGCTATG GCTTTGTGCA TTAAAAATGG CTTTCTTGCG 60 GCTGCCCTTGTACTTGTTGG GCTGTTAATG TGCAGCATCC AAATGATAGG GGCACAATCT 120 ATTGGAGTATGCTATGGAAA AATTGCCAAC AATTTACCAT CAGAACAAGA TGTCATAAAC 180 CTATACAAGGCTAATGGCAT TAGAAAGATG AGAATCTACT ATCCAGATAA AAACATCTTC 240 AAAGCTCTCAAAGGAAGTAA CATTGAGATC ATTCTTGATG TTCCAAATCA AGATCTTGAA 300 GCCCTAGCCAATTCTTCAAT AGCCAATGGT TGGGTTCAAG ATAACATAAG AAGTCATTTC 360 CCATATGTTAAATTCAAGTA CATATCTATA GGAAATGAAG TATCTCCCAT AAATAATGGT 420 CAATATTCACAATTTCTTCT TCATGCAATG GAAAATGTGT ACAATGCATT AGCAGCATCA 480 GGGTTGCAAGATAAGATCAA GGTCACAACT GCAACATATT CAGGGCTCTT AGCAAACACC 540 TACCCACCCAAAGCTAGTAT ATTTCGAGGA GAATTCAATA GTTTCATTAA TCCCATAATC 600 CAATTTCTAGCACAAAATAA CCTTCCACTC TTAGCCAATG TCTACCCTTA TTTTGTTCAC 660 ATTTCCAACACTGCTGATGT CCCACTTTCT TATGCATTGT TCACACAACG AGGAAAAAAT 720 TCAGCAGGGTATCAAAATCT TTTTGATGCC ATTTTGGATT CTATGTATTT TGCTGTAGAG 780 AAAGCTGGAGGACCAAATGT GGAGATTATT GTATCTGAAA GTGGCTGGCC TTCTGAAGGA 840 AACTCTGCAGCAACTATTGA AAATGCTCAA ACTTATTACA GAAATTTGAT TGATCATGTT 900 AAAAGAGGGGCAGGAACTCC AAAGAAACCT GGAAAGTCTA TAGAAACTTA TTTATTTGCC 960 ATGTTTGATGAAAATGTTAA GAAAGGAGAA ATCACAGAGA AACACTTTGG GCTCTTTTCT 1020 CCTGATCAGAGGGCAAAATA TCAACTCAAT TTCAATTCTT TGATGCCAAT ATATATTGAT 1080 ATATCTAGAGTGATATGAGT AATAAGGAGA ACTGTTATGT TTTTCTCCAA TTGAAAATGT 1140 AACTATGGTTTCACTTTGAT ATCTATATGT CATATTTATT GAAATCACGT CTTTTGGTTT 1200 T 1201 (2)INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1181 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 27: GGTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTAGGAAGCAAGGAAG 60 ATTTACTCTT ATTATACAGT AGTATGGAGA TCACATTCTA TTAAACTGATACAGACTATG 120 TTTTCACAGG CACCAACACC ATTAGTTTGT TCCAAGTACA GGGCTGAGCTTAACGGATAG 180 AACTCTCTCT GGAAATGAAG ATGACAGCAG ATGACAGTCG AATTCGGAGTCAAAAGAGCT 240 ATCGAAACTG ATATTCGTGC TGGTGCTAAA CTCATTCGCT TCCATTTCCACGATTGTTTC 300 GTCCAAGGCT GCGATGGGTC TGTTTTGCTA GAGGATCCTC CTGGCTTCGAGACCGAGCTC 360 AACGGACTTG GAAACTTAGG AATCCAAGGA ATCGAGATTA TCGACGCTATCAAAGCTGCC 420 GTCGAGATAG AATGCCCTGG CGTTGTCTCC TGTGCCGACA TCCTAGCTCAAGCCTCTAAG 480 GACTCTGTCG ACGTGCAAGG AGGACCCAGT TGGAGAGTTC TATACGGCAGAAGAGATAGC 540 AGAACAGCCA ATAAAACAGG GGCTGATAAC CTCCCAAGCC CCTTCGAAAATCTTGACCCA 600 CTCGTAAAAA AATTTGCAGA CGTTGGTTTA AATGAAACCG ACCTTGTTGCTCTATCAGGG 660 GCACATACGT TTGGTCGGTC CAGATGCGTG TTCTTCAGTG GTCGTTTGTCCAACTTCAGT 720 GGCAGTGGGC AACCAGATCC AACGCTGGAT CCAACTTACA GGCAAGAACTTCTAAGCGCT 780 TGTACAAGCC AAGACACACG AGTGAATTTC GATCCAACAA CACCTGATAAATTTGATAAG 840 AATTACTTCA CCAATCTTCG AGCCAATAAA GGGCTGTTAC AGAGTGACCAAGTTCTGCAT 900 TCAACGGAAG GGGCTAAAAC AGTTGAAATT GTTAGACTTA TGGCGTTGAAACAAGAAACT 960 TTCTTTAGAC AATTTCGGTT GTCGATGATT AAGATGGGCC ACATTAAACCATTAACTGGA 1020 AGCCAAGGGC CAATTAGAAG AAACTGCAGG AGGGTTAATG ACTTGGGAAGTGAAACAGGG 1080 CATGATGTTA TGTAAATTTT GTCTTCCCTC TTACGTTTGT TTGTTTCTCTCTTCCACTTC 1140 CATCTCCTTT CTATAATAAA TTAGCTCCAC TACATCACCT C 1181 (2)INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:686 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 28: AATACACAGA TTAATATATG TATCTAAAGA AGATGATGAA TGGAGGCTATAGTTTATTTT 60 TTATTTTAAA AAAAAAAAAT TCAGCCACCG ATTTGCGAAT TTCAACAATACAGGAAGGCC 120 AGACCAATCA TTGAACCCAG ACTACAGGAG CTTTCTTGAA GGGGTTTGTTCAGCAGGAGC 180 AGACACAAGA GCGAATTTCG ATCCAGTAAC ACCGGATGTG TTTGACAAAAATTACTACAC 240 AAATCTTCAA GTGGGGAAGG GGCTTTTGCA GAGCGATCAA GAGCTGATCTCAACACCTGG 300 AGCTGACACC ATCGTCATTG TTAACAGCTT TGCAGAAAGA GAAGGAACATTCTTCAAGGA 360 GTTCAGACAG TCGATGATCA ATATGGGAAA TATAAAGCCA TTGACTGGTGGACAAGGGGA 420 AATTAGAAGA AACTGCAGGC GGGTTAATTC AAACTCTGGT TTGTTGGGTGGAGAAGGAGA 480 AGGATCAGAA GGCCACGATG TTATGTAAAA CTAAAAAAAT GAGCTGCCTGTTTCTGCATA 540 TGTGGTGTTC ATCATATCCA TAACTTATAA TTAAGCATAA TGTGTGTGTTAATTTAGTTG 600 GGTGTTTGTG CTTTCTTCCA TGAATAAAAA TGCAGGGGTA GGCTCATCTTTTGTTTTGAT 660 TAAGCTTACT TATTATGTTG TCGGAT 686 (2) INFORMATION FOR SEQID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1064 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AAACAATGAACATTAAGGTA TCATTACTTT TCATTCTACC AATATTCTTG CTTCTCCTAA 60 CCAGCAAAGTAAAAGCTGGA GATATTGTAG TCTATTGGGG CCAAGATGTA GGAGAAGGTA 120 AATTGATTGACACATGCAAC TCTGGTCTCT ACAATATTGT CAACATTGCC TTTTTATCTT 180 CTTTTGGCAATTTCCAAACT CCTAAACTTA ACTTAGCTGG CCATTGTGAA CCATCTTCTG 240 GTGGTTGCCAACAGTTGACA AAAAGCATCA GACATTGTCA AAGCATAGGC ATTAAAATCA 300 TGCTCTCCATTGGAGGTGGA ACTCCTACCT ACACATTATC CTCAGTTGAT GATGCCAGAC 360 AAGTTGCTGATTACCTGTGG AACAATTTTC TCGGCGGCCA ATCATCTTTT AGGCCACTTG 420 GAGATGCTGTATTAGATGGC ATAGATTTTG ATATTGAACT TGGCCAACCA CATTATATTG 480 CACTTGCCAGGAGACTTTCA GAACATGGCC AACAAGGTAA AAAATTATAC TTAACTGCAG 540 CACCACAATGTCCTTTTCCT GATAAACTTC TTAATGGTGC ATTGCAAACT GGTTTATTTG 600 ACTATGTTTGGGTCCAATTT TACAACAATC CCGAGTGCGA GTTCATGAGC AATTCAGAAA 660 ATTTCAAGAGGAGGTGGAAT CAGTGGACAT CAATCCCTGC AAAGAAGTTG TATATTGGAC 720 TTCCAGCAGCCAAGACAGCC GCGGGTAATG GCTATATTCC AAAGCAAGTG CTAATGTCAC 780 AAGTTTTACCATTTCTAAAG GGGTCTTCAA AATATGGAGG TGTCATGCTT TGGAATAGAA 840 AATTTGATGTCCAATGTGGC TATAGCTCTG CTATCAGGGG TGCTGTTTAA GTTCTGAATG 900 AACAAGGCGCCCCTGAATCG CTATAAGCCA TCGTTAAGGC CTAAATAAAG CAAGTTAATT 960 TGCTGTTATCTGCCTAGAAA GTACTTAAGT TTTAATTTGT ACTGATGAAA ATGTGAAGGT 1020 CATCTTGTTTCCTTCTTGAT AATAGTAGTA CTATGGTTCT CTTT 1064 (2) INFORMATION FOR SEQ IDNO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1018 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TGAAGTGTTAGCACACACAC ACAAAAAAAA CTTAAAGTTA TGATCAAATA TAGTTTTCTT 60 TTGACAGCATTAGTGCTATT TCTTCGAGCA TTAAAACTAG AAGCAGGGGA TATAGTAATA 120 TATTGGGGCCAAAATGGGAA TGAAGGTAGC TTAGCTGACA CTTGTGCAAC AAATAACTAT 180 GCCATTGTCAATATTGCTTT CCTTGTAGTT TTTGGGAATG GCCAAAATCC AGTGCTAAAT 240 TTAGCTGGTCATTGTGATCC AAATGCTGGT GCATGCACTG GCTTAAGCAA TGACATTAGA 300 GCTTGTCAAAACCAAGGCAT CAAAGTTATG CTTTCTCTTG GTGGTGGTGC TGGAAGCTAT 360 TTTCTTTCTTCTGCTGATGA TGCTAGGAAT GTGGCAAATT ATTTGTGGAA CAATTATCTT 420 GGAGGTCAATCAAACACACG TCCACTAGGA GATGCAGTTC TAGATGGAAT TGATTTTGAT 480 ATAGAAGGCGGGACAACACA ACATTGGGAT GAATTAGCAA AAACTCTATC ACAATTTAGC 540 CAACAAAGGAAAGTATACTT AACTGCAGCT CCACAATGTC CATTCCCAGA TACATGGTTA 600 AATGGGGCACTTTCCACTGG CTTATTTGAT TATGTTTGGG TTCAATTTTA CAATAATCCA 660 CCGTGTCAATACTCCGGTGG GAGCGCGGAC AATTTAAAAA ATTACTGGAA TCAGTGGAAC 720 GCGATTCAAGCTGGAAAAAT TTTTCTGGGA TTGCCAGCAG CTCAAGGAGC AGCTGGAAGT 780 GGTTTTATACCATCTGATGT TCTTGTTTCT CAGGTTTTAC CATTAATTAA TGGTTCACCA 840 AAGTATGGGGGTGTTATGCT TTGGTCTAAA TTTTATGACA ATGGTTATAG CTCTGCTATT 900 AAGGCTAATGTTTGAGATAT ATGATCATAG CTAGTCAGCT TGTATTAATA TGATGACGTC 960 AATAATGTTATATTATAAAC TATATAGTAC TCAATAATAA GGCTTTGAAA GTTACTTA 1018 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:645 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 31: AAAATGGAGA GAGTTAATAA TTATAAGTTG TGCGTGGCAT TGTTGATCATCAGCATGGTG 60 ATGGCAATGG CGGCGGCACA GAGCGCCACA AACGTGAGAT CGACGTATCATTTATATAAC 120 CCACAGAACA TTAACTGGGA TTTGAGAGCA GCAAGTGCTT TCTGCGCTACTTGGGATGCC 180 GACAAGCCTC TCGCATGGCG CCAGAAATAT GGCTGGACTG CTTTCTGTGGTCCTGCTGGA 240 CCTCGAGGCC AAGATTCCTG TGGTAGATGC TTGAGGGTGA CGAACACAGGAACAGGAACT 300 CAAACAACAG TGAGAATAGT AGATCAATGC AGCAATGGAG GGCTTGATTTAGATGTAAAC 360 GTCTTTAACC AATTGGACAC AAATGGAGTG GGCTATCAGC AAGGCCACCTTACTGTCAAC 420 TATGAATTTG TCAACTGCAA TGACTAATTA ATCTGCTTCC AGATATATAAGTACCATCAT 480 AAAAAACCAC AATAATTCAT ATATACTGGC ATATCTTATT TTTAAGAGCCGTTTAGAATA 540 AGAAGGGGCT GAGCTAGCTT TTAATGTGTA TGGTATTATC TGAAGCTCTACATGCCCTTC 600 ACTATTATAG ATAAAAGCTT GTTCTTGTTG TTAAAAAAAA AAAAA 645 (2)INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:635 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 32: AAAATGGAGA GAGTTAATAA TTATAAGTTG TGCGTGGCAT TGTTGATCATGAGCGTGATG 60 ATGGCAATGG CGGCGGCACA GAGCGCCACA AACGTGAGAT CGACGTATCATTTATATAAC 120 CCACAGAACA TTAACTGGGA TTTGAGAGCA GCAAGTGCTT TCTGCGCTACTTGGGATGCC 180 GACAAGCCTC TCGCATGGCG CCAGAAATAT GGCTGGACTG CTTTCTGTGGTCCTGCTGGA 240 CCTCGAGGCC AAGATTCCTG TGGTAGATGC TTGAGGGTGA CGAACACAGGAACAGGAACT 300 CAAGCAACAG TGAGAATAGT AGATCAATGC AGCAATGGAG GGCTTGATTTAGATGTAAAC 360 GTCTTTAACC AATTGGACAC AAATGGATTG GGCTATCAGC AAGGCCACCTTATTGTCAAC 420 TATGAATTTG TCAACTGCAA TGACTAATTA ATCTGCTTCC AGAAAATAAGTAGCTACTGT 480 AGTATCTTAT TTTTCAGAGC TGCGCTGTTT AGAATAAGAA GGGGGCTGAGATTGCTTTTA 540 ATAGTATACT GTAGGTATTA TCTGAAGCTC TACATGTTTG GTTGCCCTTCACGATTATAG 600 ATAAAAGCTT GTTCTTAGTA CTAAAAAAAA AAAAA 635 (2)INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:860 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 33: GGGAACAAAA GCTGGAGCTC CACCGCGGTG GCGCCGCTCT AGAACTAGTGGATCCCCCGG 60 GCTGCAGGAA TTCGGCACGA GCAACTTAGA AAAATGAATT TTACTGGCTATTCTCGATTT 120 TTAATCGTCT TTGTAGCTCT TGTAGGTGCT CTTGTTCTTC CCTCGAAAGCTCAAGATAGC 180 CCACAAGATT ATCTAAGGGT TCACAACCAG GCACGAGGAG CGGTAGGCGTAGGTCCCATG 240 CAGTGGGACG AGAGGGTTGC AGCCTATGCT CGGAGCTACG CAGAACAACTAAGAGGCAAC 300 TGCAGACTCA TACACTCTGG TGGGCCTTAC GGGGAAAACT TAGCCTGGGGTAGCGGTGAC 360 TTGTCTGGCG TCTCCGCCGT GAACATGTGG GTTAGCGAGA AGGCTAACTACAACTACGCT 420 GCGAACACGT GCAATGGAGT TTGTGGTCAC TACACTCAAG TTGTTTGGAGAAAGTCAGTG 480 AGACTCGGAT GTGCCAAAGT GAGGTGTAAC AATGGTGGAA CCATAATCAGTTGCAACTAT 540 GATCCTCGTG GGAATTATGT GAACGAGAAG CCATACTAAT GAAGTAATGATGTGATCATG 600 CATACACACG TACATAAAGG ACGTGTATAT GTATCAGTAT TTCAATAAGGAGCATCATAT 660 GCAGGAYGTA TCAATATTTA TCAAATAATA CAAATAAGAG CTGAGATTACGAGAATCTAT 720 TTAAATTAAA AGTTACATAC TTAATTATTA TAGTTATATA TGTAAAATATGTGGCCTTTT 780 TAAAAGTTAC ATAATTAATT ATTATAGTTA ATGTCTTTCA AAAAAAAAAAAAAAAAAACT 840 CGAGGGGGGG CCCGGTACCC 860 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 783 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GAAGATCAGACTTAGCATAA CCATCATACT TTTATCATAC ACAGTGGCTA CGGTGGCCGG 60 ACAACAATGCGGTCGTCAAG GCGGTGGTCG AACTTGTCCC GGTAACATCT GCTGCAGTCA 120 GTACGGTTACTGTGGTACCA CCGCGGACTA CTGTTCTCCG ACCAACAACT GTCAGAGCAA 180 TTGTTGGGGAAGTGGGCCTA GCGGACCAGG GGAGAGCGCG TCGAACGTAC GCGCCACCTA 240 CCATTTCTATAATCCGGCGC AGAATAATTG GGATTTGAGA GCCGTGAGTG CTTATTGCTC 300 CACGTGGGATGCTGATAAGC CGTACGCATG GCGGAGCAAG TATGGCTGGA CCGCCTTCTG 360 CGGGCCGGCAGGACCTCGTG GTCAAGCTTC TTGCGGCAAG TGTTTAAGGG TGAAGAACAC 420 AAGAACAAATGCTGCAGTAA CTGTGAGAAT AGTGGACCAA TGCAGCAACG GAGGCTTGGA 480 TTTGGATGTAGCAATGTTCA ATCAAATAGA CACCGATGGT TTTGGCTATC AACAAGGCCA 540 TCTCATTGTTGACTACCAAT TTGTCGACTG TTGGCAATGA GCTCATTGGG CAGCCTGATT 600 CCAGAAACATGCTTGTTTCG GCCATTGATC GCGTTTGATA TTATGTAATG ATTTTGAGGT 660 CAATATCGATCGGTCTACAT AAAAATAATA AAGACCGCTA TATATGTATT GTCGAGGGAT 720 ATATGTTTCGTATCAATAAG GAAATTTTAA ATATTATTAT CAAAAAAAAA AAAAAAAAAA 780 CTC 783 (2)INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:979 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 35: GGCACGAGCT CGTGCAATTC GGCCGAATTC GGCACGAGAA AATATGGCAAATATCTCCAG 60 TATTCACATT CTCTTCCTCG TGTTCATCAC AAGCGGCATT GCTGTTATGGCCACAGACTT 120 CACTCTAAGG AACAATTGCC CTACCACCGT CTGGGCCGGA ACTCTCGCCGGTCAAGGACC 180 CAAGCTCGGC GATGGAGGAT TTGAATTGAC TCCAGGTGCT TCCCGACAGCTCACGGCTCC 240 TGCAGGATGG TCAGGCCGGT TCTGGGCTCG TACAGGCTGC AACTTTGACGCCTCCGGAAA 300 CGGTAGATGT GTAACCGGAG ACTGTGGCGG TCTAAGATGT AACGGCGGCGGAGTTCCTCC 360 CGTCACTCTG GCTGAATTCA CTCTAGTAGG CGATGGCGGC AAAGATTTCTACGATGTGAG 420 CCTCGTAGAT GGTTACAATG TCAAGCTGGG GATAAGACCA TCCGGAGGATCGGGAGATTG 480 CAAATACGCA GGCTGTGTCT CTGACCTCAA CGCGGCTTGC CCCGACATGCTTAAGGTCAT 540 GGATCAGAAC AATGTCGTGG CCTGCAAGAG TGCCTGTGAG AGGTTTAATACGGATCAATA 600 TTGCTGCCGT GGAGCTAACG ATAAGCCGGA AACTTGTCCT CCCACGGACTACTCGAGGAT 660 TTTCAAGAAC GCTTGCCCTG ACGCCTATAG CTACGCTTAT GACGACGAAACGAGCACCTT 720 CACTTGTACC GGAGCTAACT ACGAAATCAC TTTCTGCCCT TAAAAACCGAAGCTTCGAGT 780 TAGATACAGT CGGGTTTAAT TATCTCTCAC GTTTCTTTTG CTTATTATGTACGGAAAGAT 840 AAATAAGGAA AGCTGATGAC TATGAATCAT CGTCTTCCAC TTTTAAGCTTTTTTAGTGAG 900 TATTAGTCAG TTGTTACACT CAGCTGATTT GTTTACAAAG AAATAAAACAAAATGATTGA 960 TCTAAAAAAA AAAAAAAAA 979 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12124 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Cucumis sativus(C) INDIVIDUAL ISOLATE: Cucumber Chitinase Genomic DNA (vii) IMMEDIATESOURCE: (B) CLONE: pBScucchrcht5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GAATTCTCTT TTTACAATTA CAAAACTAAA ACGAACATTG TATTTTAAAA TTTAAATAAT 60TAAAATGAAT ACATTACAAG TATAGATATC AAAATAAAAA AATCAAAATA GGATTAAAAT 120TGCTCAGCTT ACATAAACTT ATATTGTAGA CTTTAGAGTT GAGGTTCGAT ATCTCACGTT 180ACATGCTGTG GTGTATATAT ATATTTATGA AGCATATTTT GTCACTATTA TAAGCTTTTT 240ATTTTTGAGG CTAAATATTG CAAGTGGAGG AGGAAATTAG TTAAGCACAT AACAAAATCG 300TAGTTGGCCA TTACATGTTG CGAGAAATTC GATGTGAAGA TCATTATTAT ATATTCAACT 360CTTCAAATTA TTATAGATTC CTTGACTATT TCTCTTTCTA TTTTATATTA TTACATCAAT 420CCCTCAACAA ATAAATCATT CAACTATACA AATTTAATAA TATGTGTATG GAAATGATCA 480TCTCATCATA ATTAAATTAA ATTTAAAACT ATGAACACCA ACACCAATCA ATAAACCGTA 540CATAACTCAA ACTATTATTA TTAAATCACT TAATTTAGTC ACAAACTAAC GTTTATAGAC 600ATGGTTGGAT TCACAAGGAA TAACATTCTT CCTATTTTAA AAAAAAGTTA GTACTTATGT 660TTGCTTGTAT ACCAAACACG TGTATTAATT AATGTCAAAT TACTTACATA ACAGCTATAA 720ATAATACTTA CGATCGACTA ATTAATAATT AAAAATAATA TAATGGTATC TAGAAATAGA 780TCGATTATAG ATGTTATGTC TAGAGTTGGT CCTTATAGGT GATTGGGTGA TCGAGGTATT 840CACACATTAC ACATAAGTAA TGAGTGGACA AAAGTTGGTC CTTGAGGTGG TCGTCGATTG 900TTTAGAGTTG ATTGGGCACA AAGGTTTCAC CTAAAGTATG GGTTTACCAA AGATGTGACC 960TAAAACGATG GGTGTTGGTA GCCATCACTC AAACCATTTT AGTAAACTTA AATAATTATA 1020TAAATTAGGT TAACATCAAG CAATCAAAAA TCGTGCTTTG TATTTTAAGT GAATTTCTTG 1080AAATGATTAT CTCTTATCAA CGTATTTCAT GACAAACTCA TTTTTCTTAT AAATTATTTC 1140TCTATTAGTT ACCTCACACT TTAACGTGTT TAAAATAATT TTTCTCACAC AATTAAATAC 1200TTAAAATCCT AAGCTAAATA CAATTAAATA TAATCTTGCA ATTTCAAAAT AATTAAAATC 1260ACTGGAAAAA AAAAAGGAGA AACTATTGAT AGTCCACATT AATCATAAAT TTAACTATAT 1320GAAATTAAAA AGTTATTATA AAATATCCTT CAAATTACCT CATGCATAGT AAATTTTTTT 1380TATAATTTTC TTTCAGAAAG TTCGATCAAG TCATAAGATT ATCTCTACAA AATAAGTATA 1440AGTTAATGAG TAACCTAAAA TGCAGATTTG TTGAAGAAAA AACAAAATTA CTGTACTGTG 1500TGTTTGTAAA CTTTTCCACA TATATATACA GCTATTTGTG ACAATGATAT AATGTGAACG 1560TGTGGAATAA TTTGTTTTTG ATAGAAGTTG GAGTTTGAAA TGTCTAACTT TTAACCAAAT 1620AAATTCCGTT AATTACGGTG ACTTAGGACT CACCTTAACT ATATAGTCAA TAGGTATTTT 1680CTTTTGTTCA CACAACTTTT TTAATATACT CTTTTACGTA AGTAATGTAA CATAAACTAT 1740CGCTGCAAAA AGAACAGGCT TTGCTCGCCT AAAAGCACGT CGGCATATTC ATCTCTGTCA 1800GTAGACAAAA ATTCTGTCAG CAGAAACTCG TCGGAGTTGA TCTTCCAACA ACAAGACGAG 1860GTCGTCCGCT GTTAGAAAAA ATGTTGATGG TTTAAATATA TTGTCTGCAG TAGTGATAAG 1920CAGACTAATT GTTATTAGAG GGTTATAGAG GTTGAAATTC TTACAAATTT TCTAATCGTC 1980AAACTAATTG AGAGTTTAAA GAGTTTCTCA TAATCTTCAA AGGATGGGTA GGAATTTTTT 2040GAGTACCTAA TAAGTTATAA GCAAAGATGG TTGATTGTGC TGGGATTAAA TTACAAATTT 2100AGTACAAAAA TATTCATATT AAAGTATAGT TCCATTTGGT TCTTTCACCT TTAGTTTTGT 2160GTCATAATCT TCAAACTTTT AACAATCCAA GGCAACTACT AAACAAAATT TAATCTGTTG 2220AATAACAAAA CTACAAATTT TTTTAAAAAA TATCGGAGAA TAAACTTATA ACTATCACAA 2280AACTCTCGTA CTTAACACCA CATAATCATA ATCGTATTCT CCATGAAAAT TTCAAATCAA 2340CCATTTTTTT CTCTCTTCAA TTAGAATGAT CGAACAAGCC AATTCATTAC ATAATTTGTA 2400ACATTTTTTT CCAAACCCAA ATGACACTCT ACAAATACTT TGATTTGATC AACAATAACC 2460CTACGTGATT ACCTTTTCCC TTCCCAATAA ATTCACTTCA TATTTTCCAC TGTTTAAACA 2520CATAATCTCA AAGGAAAAAG CTCTTTAAGA AATGGCTGCC CACAAAATAA TAACTACAAC 2580CCTCTCCATC TTCTTCCTCC TCTCCTCTAT TTTCCGCTCT TCCAACGCGG CTGGAATCGC 2640CATCTATTGG GGCCAAAACG GCAACGAAGG CTCTCTTGCA TCCACCTGCG CCACTGGAAA 2700CTACGAGTTC GTCAACATAG CATTTCTCTC ATCCTTCGGC AGCGGTCAAA CTCCGGTCCT 2760CAACCTTGCC GGTCACTGCA ACCCTGACAA CAACGGTTGC GCCTTTGTGA GCGACGAAAT 2820AAACTCTTGC CAAAGTCAAA ATGTCAAGGT TCTCCTCTCT ATTGGAGGCG GCGTAGGGAG 2880ATATTCACTC TCCTCCGCCA ACAATGCGAA ACAAGTCGCA GGCTTCCTCT GGAACAACTA 2940CCTCGGCGGG CAGTCGGATT CCAGGCCACT CGGCGATGCG GTTTTGGATG GCGTTGATTT 3000TGTTATCGGG TTTGGCTCGG GCCAGTTCTG GGATGTACTA GCTCGGGAGC TAAAGAGTTT 3060TGGACAAGTC ATTTTATCTG CCGCGCCACA GTGTCCGTTC CCAGACGCTC AGCTAGACGC 3120CGCGATCAGA ACTGGACTGT TCGATTCCGT CTGGGTTCAA TTCTACAACA ACCCGCCATG 3180CATGTATGCA GATAACGCGG ACAACCTCCT GAGTTCATGG AATCAGTGGG CGGCGTATCC 3240GATATCGAAG CTTTACATGG GATTGCCAGC GGCACCGGAG GCAGCGCCGA GCGGGGGATT 3300TATTCCGGCG GATGTTCTTA TTTCTCAAGT TCTTCCAACC ATTAAAACTT CTTCCAACTA 3360TGGAGGAGTG ATGTTATGGA GTAAGGCGTT TGACAATGGC TACAGCGATG CCATTAAAGG 3420CAGGATCCTA TTGAAAAAGA GTAGCTATTG TTATGGAGTA AGGCGTTTGA CAATGGCTAC 3480ACACCTGCCT CTCTCACTTG AAATAGAGCA GGTATTGGTT TGAAATCAAT TATAACCAGA 3540GCCCATCAGT ATACTCCGTC CTAATTAATC TCAAGTTGTC AAAATTATTA GAAGCAAATT 3600TCTAATCAAA CAACAATATT ACTCTATCCT TACACGGAAG AATGCTCAAG GAGTTTACCG 3660GAACTATCTT TTACTCTGAA ACCGTCTCCA AACCACTACT ACATCAATAA AACCATCTCT 3720AACTCTAGAA AACAACAGAA TTGCCCATTC AACTCTACAG TGAACGAATC CAAGACATAG 3780TTTCCAAAAA GTTCAAGAGT TAATTAATTA AAGATTTGGT CAAGTCCAAT CAAAATAGAG 3840CTTGAATATA TAAAAGAAAA TAATCATACC CTTGATATTG TTGTATTCAA GAAGGGAAAC 3900GTAGACGGCC ATATCAACCT TTGGGTACTT GGCAAAAGAA AAGAAAATAT AGTAGACTAG 3960ATAATTGAAT TTGTAGTTAG ACTTGTAAAA TAGTAAGGAG CAACTTTGTA GATATATCTT 4020TACAAAGGTG TGGTAGAGGC AACAACCATT TCTTTATCTT CTTATAAATC AACTTGTGGA 4080TCGAAGTTTA ATATCTTGAA CCGTAATAAA ATCATAAGTT TAACTATTTG AAGTCTTAAA 4140AAACTTATTA TAAAATATCC TTCAATTTAT CTAATGCATA GTAGAAGTTA TAAGATTCTC 4200TCTAAAAATA AGTATATAAA TTAATGAGTA CCCTAAAATG CAGGTTTGTT GAAGAAAAAA 4260TAAAATTAGT ACTGTTTGTA AACTTTTCCA CATATATACG CAGCTACAAT GTTAACGTGT 4320GGAATAATTT GTTTTGGATA GAATTTGGAG TTTGAAATGT CTAACTATAA TCAAACAAAT 4380TCCATTAATT ATGATGACTT AGACTCACTT TAACTATATG TAGTCAATAG ATATTTCTAG 4440ATTTCACGGC TAATTTAATT GAATTTTGGA CTTTTTTAAT GTACTCTTTT ACATATGTAC 4500GTAATGCATA ACTATTGCTA CAAAAAATAG GTTTCTCTCG CTGCACAAAA CAGTCGATAA 4560ATACCTAAAA GCAGTGGATA AAAACTTGTT GGGAGTTCAG TTGGAAGAAA CTCGTCGGGA 4620CTTGATCTCC TGACAACAAA GCAAGGTCAT CAGTTGTTAG AAAAATTGCA AATGGTGTAA 4680ATACGTTGTC GACGGTAGTG ATATATCGTC CAACCCTTAC ATTACGTCGT TGGCGGTTGT 4740ACTATCTCCA GTGCACCCCT TGACCGGTTG ACAATTATCC ACAAAACACA CCAATGTTAT 4800TCTAGCTAGT TAAATCTCCC AACATTACAT AAAAAACAAA ATGTGTTTGA CACAAATGTG 4860AAAATAAAGA TTAATTGAAC AGTACATATG ATCTTAAATC AAATTCAAGA GACGATATGT 4920ATAGTCATAT AAACTCTATA CGTAACATAG CTATACACTT TTTCGGCTAA CTTGAACTTA 4980GTTAAACAAG TAAAATAAGT TGATCATGTA CCATGCTTAC TATTTAGAAG GTCACAGAGG 5040TTCAAATACT TGCAAAAAAT TTTAACGTTG AACTTTTAAA ATAAATAAAT TTCATTGTTA 5100TACCTTAAAC TAATTGGAGT TAAAGTGTGA TCTCTAGGTT AAATTACAAT TTTAGTACAA 5160AAAAAAATTA TATTCGAGTC CAGCCATCTA GTTCTTCCAT TTTAGTTTTG TGTCATAATC 5220CTAAACTTTC AATTTTATAT TTAATAAATT ATTGTATTGG ACTTAACAAA TCATAACTCA 5280ATTATTGTTT TATCAACTTC AAAATTAACG TTTGGTTTTC CTATCACACA TTATCAAAAA 5340GAGAAACGTT CACGTTCAAC AACACAATTA TAATAATAAC ATGCATCAAT TATTAAAATT 5400TCACAAACCC ACAAAAGAAA AAACAACAAC AACAAAATTG AAATTAAGTC CAGAGGTCCT 5460TCCATATACC TAAACCTCAA TTTTACTTAT AAACATTAGT TAACATTTTA AATATCTAAT 5520AATCCAACCA TATGACATAT TAGAGATTTA TGGACTTATT AAGCACATGT TTAACAATAG 5580TTCAAAGGCC GCCCTACTAA TAACATATAC AAATTTAATT TGTTGAACAT AACTACAATT 5640TTTTTTAAAA AAATATTAGA GAATAAACTT ATAATTTAAC AATATTTTAA TCACATAGCT 5700TATAATAAAC TTAATTATAA TCACAAAAGT CTAGTACTTA TATATAATTT GTAGAGATAT 5760GTTTACTTTG ACCTTGACTC CACGTAATCG TATTTCCATG GAAATTCAAA TTAATCAACC 5820ACTTTTTTTC TCTCTTCAAT TAGAACACGG CAATTGATTA AATAATTTGT AACATTTTTT 5880CCAAATCCAA ATGACACTTC CAAAATTATA TTATATGATC TTATACTTTG ATTTGATCAA 5940CAATAACCCT TCGTGATTGC CTTTTCCCTT CCCTATAAAT TCACTTCACA TTTTCCATTG 6000TTTAGACACA CAAACTCAAA GAAAGCTCTT TAAGCAATGG CTGCCCACAA AATAACTACA 6060ACCCTTTCCA TCTTCTTCCT CCTTTCCTCT ATTTTCCGCT CTTCCGACGC GGCTGGAATC 6120GCCATCTATT GGGGTCAAAA CGGCAACGAG GGCTCTCTTG CATCCACCTG CGCAACTGGA 6180AACTACGAGT TCGTCAACAT AGCATTTCTC TCATCCTTTG GCAGCGGTCA AGCTCCAGTT 6240CTCAACCTTG CTGGTCACTG CAACCCTGAC AACAACGGTT GCGCTTTTTT GAGCGACGAA 6300ATAAACTCTT GCAAAAGTCA AAATGTCAAG GTCCTCCTCT CTATCGGTGG TGGCGCGGGG 6360AGTTATTCAC TCTCCTCCGC CGACGATGCG AAACAAGTCG CAAACTTCAT TTGGAACAGC 6420TACCTTGGCG GGCAGTCGGA TTCCAGGCCA CTTGGCGCTG CGGTTTTGGA TGGCGTTGAT 6480TTCGATATCG AGTCTGGCTC GGGCCAGTTC TGGGACGTAC TAGCTCAGGA GCTAAAGAAT 6540TTTGGACAAG TCATTTTATC TGCCGCGCCG CAGTGTCCAA TACCAGACGC TCACCTAGAC 6600GCCGCGATCA AAACTGGACT GTTCGATTCC GTTTGGGTTC AATTCTACAA CAACCCGCCA 6660TGCATGTTTG CAGATAACGC GGACAATCTC CTGAGTTCAT GGAATCAGTG GACGGCGTTT 6720CCGACATCGA AGCTTTACAT GGGATTGCCA GCGGCACGGG AGGCAGCGCC GAGCGGGGGA 6780TTTATTCCGG CGGATGTGCT TATTTCTCAA GTTCTTCCAA CCATTAAAGC TTCTTCCAAC 6840TATGGAGGAG TGATGTTATG GAGTAAGGCG TTTGACAATG GCTACAGCGA TTCCATTAAA 6900GGCAGCATCG GCTGAAGGAA GCTCCTAAGT TTAATTTTAA TTAAAGCTAT GAATAAACTC 6960CAAAGTATTA TAATAATTAA AAAGTGAGAC TTCATCTTCT CCATTTAGTC TCATATTAAA 7020TTAGTGTGAT GCAATAATTA ATATCCTTTT TTTCATTACT ATACTACCAA TGTTTTAGAA 7080TTGAAAAGTT GATGTCAATA AAAACATTCC AAGTTTATTT AAATTTTGTG TAAACTGTTT 7140GAAGTTTAAA TACAATATAA TCTCATTAAC GTAAGAATTT GATATTTTAG CCAAATTTTT 7200AAATCGATCC TCTGTCTTCT TTCTAGTTAA TTATATATCA ATTTTATTTC TTACTTGGGT 7260GAAATTTTTT TCTAATTAAA AACAATAGTA CATACAATAA GTTTGATATA ATCACTAATT 7320CAATCTTAAG CTTTAATAGA TGAAGTTAAA TTTGATATTA AATCTAACAA TTTATGTTAT 7380CGGTTACTGT TGAAAGAGAT GAAATTATCA AAATAAATGG AGTTGAAGAT TAATTAATCA 7440AATCATTGAC GTAGACGTTA CTGTGATTGT TTTAAGTTTA CAAATATATT GACAGTCAAC 7500TATTTTCCTA ATTCTAAGAT AATCAAACTT GTTTAATTCC TAAAGAATCG AAAGAAAAGA 7560AGTAATAAAA AAACTTGTTT AATTCAATCT TTAAATTGGT TTTATTTTCC AACTAGCTAA 7620ATTATTCATC TTATTATCAT TTGTATTTGT AGTCATATTA ATATTAACAT GTGATTTTTT 7680TTTAAAAAAA AATGTTTTGT TGTGTATTTA TAGAAATTGA TGCTTGAATC TTTATGATTT 7740ATACATATGT GGATAGATAG TTAAATTATT ATCTATCAAA GTTAATATAC AGAATATGAA 7800AATATGAGAT GTATTTATTT ATTGTGCAAA TGTAAATCTC GTATCTAGTT TATTAATTTC 7860TACATTCACT TTTTAGTTAA AGGGTGCAAC GAAGGATGTT TTGAGATTTC TCGAATGGTT 7920ATCATATACT TAAACTCCAT TTCCATGAAC GTACAAAGTA AATTTGAGGT AGATTGTCAC 7980TATATTCATT ATGGTTCTCA AGATAGCACA TGCATTGTGC TACATGTCAT GTTGTTTACT 8040AAGGAGCAAC TTTGTAGATG CAACAATCAT ATGATTTGTA TATATTTCAT ATTTATTTAT 8100GTTCATATGT ATTTTCCATG AAAAGTTGGA AGAACCTGAG ACTTAAGCAC GTTGGCCGGA 8160GTAAAACCAC CGCTCGGTGA AGCTGCAGAT GCCGCCTGTC GCCCCATGTA CAGCTTCTCA 8220ACCGGAAGCC CCGTCCACCA AGTTCCAAGA ATAATTCCGG AGATTGTTGA CGTTTCCATT 8280TGCATACATA CAGGGCGGAT TGTTGTAGGA TATTGAACCC AAACGAAATC AAACAAACCT 8340GTTTTAATGG CAGAAAATGG ATATGCTCTC TTCCTCACGA GGAATTTCAT TCAATAACCT 8400ATCTGCCTCA TCAAGGACCT GCTCGACATG CAAAACAGGG TAGCCAGTGT ACAAGCTTAT 8460TGTCTCTCTA TACCAAAAAA ATTTAGCCAC GGCAACATTC GATAAGGAGA TAATGATAAT 8520GTATAGAAAT TTAGATGATG TAATTAACAA AAGTACTTTA GGGAGAATCA CATGATTGTA 8580GAGGAATAAG GTGATGTGCT AAACATTATT GTATGTCATT TTCAGTACAA CTTGGGATAA 8640TATATATTTT TTATCAATGT TGAATTGTTT ATTATTGATC TTGAGATCCA TTGACGATTT 8700GTTATAATGT TTTTATTTGC TCTTTTTGAA TTGAGATCGA TTGACCCATA AACCCAAAGC 8760ATCGATAATT TTTTTTACTT GGTGGAGTCT TGGTAAAAAA GAAAATCAAG AAAATTGTAT 8820AAACTAATAT AAAAATATTT ATCTTATAAA TTAATTACTT CAAATTTGGG AGAAACTGAA 8880GATTACATAG AGGATATTTT AATGTATGGA TCTAGAATTG AAGATTACAT AGAGGATTTT 8940TTTAGTGTAT GGATGTAAAA TGTGTTGGTG TACTTAAGTT GAATTATGAA TAGAAAATTT 9000GGAGAAAGAT TATAAAAGAT TGCATAAGAT CGATTTTCGG AAAAGAGATA TTACTTAGCT 9060TGCAATTTCA ATCTTGCATT TTTATATATA TATACTTCTA AATAGACTAT CATAAGTAGA 9120ATCAATTAAC CTTTTTTTTT TTTTCATTTA GAACATTACC ATTCATTTAA ATAATTTGTA 9180ACATTTTTTC CAAATCCAAA TGACACTTAC AAAATTATAT TATATGATCT ACTTTGATTT 9240GATCAACAAT AACCCTTCGT GATTCATTTC CCTTCCCTAT AAATTCACTT CACATTTTCC 9300ATTGTTTAGA TACACGAACT CAAAGAAAGC TCTTTAAGCA ATGGCTGCCC ACAAAATAAC 9360TACAACCCTT TCCATCTTCT TCCTCCTTTC CTCTATTTTC CACTCATCCG ACGCGGCTGG 9420AATCGGCATC TATTGGGGCC AAAACGGCAA CGAAGGCTCT CTTGCATCCA CCTGCGCTAC 9480TGGAAACTAC GAGTTCGTCA ACATAGCATT TCTCTCATCC TTCGGCGGCG GTCAAACTCC 9540GGTCCTCAAC CTTGCCGGTC ACTGCAACCC TGACAACAAC GGTTGCACCA TCTTGAGCAA 9600CGAAATAAAC TCCTGCCAAA GTCAAAATGT CAAAGTCCTC CTCTCTATTG GCGGTGGCAC 9660GGGGAGTTAT TCACTCTACT CCGCCGACGA TGCGAAAGAA GTCGCAAACT TCATTTGGAA 9720CAGCTACCTC GGCGGGCAGT CGGATTCCCG GCCACTGGGC GATGCGGTTT TGGATGGCGT 9780TGATTTCGAT ATCGAGTTTG GCTCGGACCA GTTCTGGGAC GTACTAGCTC AGGAGCTAAA 9840GAGTTTTGGA CAAGTCATTT TATCTGCCGC GCCGCAGTGT CCGATCCCAG ACGCTCACCT 9900AGACGCCGCG ATCAGAACTG GACTGTTCGA TTCCGTCTGG GTTCAATTCT ACAACAACCC 9960GTCATGCATG TATGCAGATA ACACGGACGA TATCCTGAGT TCATGGAATC AGTGGGCGGC 10020TTATCCGATA TTGAAGCTTT ACATGGGATT GCCAGCGGCA CCGGAGGCAG CGCCGAGCGG 10080GGGATTTATT CCGGTGGATG AGCTTATTTC TGAAGTTCTT CCAACCATTA AAGCTTATTC 10140CAACTATGGA GGAGTGATGT TATGGAGTAA GGCGTTTGAC AATGGCTACA GCGATGCCAT 10200TAAAGACAGC ATATATCAGC TGAAGGGAAG CTCCTAAGTT TAGTTTTAAT TAAAGCTATG 10260AATAAACTCC AAAGTATTGT AATAATTAAA AAGTGAGACT TCATCTTCTC CATTTAGTCA 10320TGCTACAATT AAAATCCTTT ATTTTTACTA CAATACTATC AATGTTTTAG AATTAAAGTT 10380GATATCAATA AAAATATTCC AAGTTTATTT CAATTGTGCA AAATGTTTGA AGTACTTTAA 10440AAACAATATA ATCTCATTAA CATGAGAATT TTATATTTTA GCCATTATGT AAGAATAATA 10500TTTCTTATTT GGAAGCAATT ATGTGAGAAT TTTACTTCTT ATTTGGTTGG AATTTTTTCT 10560AATAAAAATA ATAGTATACA GGTATGTTGA TATAATCACC AATTCAATCC AAATTAAAGC 10620TTAAGTTTAA TAGATGAAGT TAAATTTTAT ATTAAATCTA ACAATTCATG TTGAACTCAT 10680AAACATATGT GATTATACAG CAAAAAAAAA AAAATTGCAA GGGTTGAGTA CCATTGTCAT 10740AGATGTCAAT ATATATATAT ATATATATAT ATATTCCATC AATATAAAAA ATTGAAAATC 10800TTTAAAATAA TATACAGAGA TGAAATTAAA CTAAATAGTA ATTAGAAGTT TTAATTTTAT 10860CCTAATGTTC TAATTTTGAT TATAAAAAAA CAACCTTGTG ACATAGCTCA ATAGTAAATA 10920AAACTGATCT CTCCATAATG TAATTAGTTG TGTTTTTAAG TCAAATAGTG ATATTCACAT 10980AACACCAAAA CACAAGAACT CATATTTTCT GATTTTTTTT TTTCTTCTCA TCAATGTTAA 11040TGTCTATTAT TACTCTTTTT TTAAGTATAA AATGTCTATG TTTGATATGA TATTCTTGAC 11100CCATTATACA CCCATAAAAA AATATATGTT TTCTAACAAC GGCAAAACTT GGAAGTATGG 11160AGTTGAGGAT TAAATTAAAT TAAAGTGTTG ATGTTGATGT TACTGTCACT ATTTTAATTT 11220TACAAATATT TGACTGACAA CTACTTTCCT TCTAAGATAA TAAACTTTTG TTTAATTGAT 11280CAATCTTCAA ATTGGTTTTA TTTAATTTCC AACTAGATTA TTCATCCTAT TCTTTGTATT 11340TGTAGTCATA TTACATATGA TTTTTATAAA AGATATATTA GAGATATCAA TAGACATTGT 11400TGAACCTTTA TGATTTATAG ATATATATGG ATAGATATTT CAAGAGTGCT CATCATATAC 11460TTAAGAATCC ATTTTTTTTA TGAACGTTCA AAGTATTGAG TAGAGATTGT CATTATATTC 11520ATTATTGTTC AAGATGGCAC CATTGTGCTA CATGTCATGT TTTTACTAAG GTTACGTTGC 11580AGATATATCT TTACAAAGGT GTGTGTTAGG GCAACAACCA TCTTTGTTAT CTTCTTCACG 11640AATTAATTGG GAGGGAGCTG GTATTAAAAA ATATGTTTTC ATGTATGATT TGTATATATA 11700TTTCCAATTC ATTATTTATG TTCATATGTA TTTTCCATGA AAAGTTATAC TCTAATAAAA 11760AGTAATTTTA TTGCATCAAA GTGATGACAT ATAGATATAT AAGATGGGCC AACAAATCCC 11820ATTTTTGTTT CCATTTCTTA TAGTTTACTT ATTCATGGAT TTATAATAAC TAATTACTAA 11880ATAAGTTTAT TGAAGAGAAG AAGCTCAAAG ACTTCCTTTA ATGGCGTTGC TGTACCCATT 11940ATCAAATGCC TTACTCCACA GCATAATTCC TCCATACTTG GAAGAACTTT TAATCCTTGG 12000AAGAACCTTA GACTTAAGCA CGTTGGCCGG AATAAAACCA CCGCTGGGTG CAGCTGCAGA 12060TGCCGCCGGT AGCCCCATGT ACAGCTTCCC AACTGGAAAC CCCGTCCACC GGTTCCAAGA 12120ATTC 12124 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1079 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 37: ACAAACCAAT AACACAAAAC ATGGCTTTCACAAAAATCTC CTTAGTCCTT CTTCTCTGCC 60 TCTTAGGTTT CTTTTCTGAA ACTGTCAAGTCTCAAAACTG CGGTTGCGCT CCAAACCTCT 120 GTTGCAGTCA GTTCGGTTAC TGTGGTACCGACGATGCATA CTGCGGTGTT GGATGCCGAT 180 CAGGTCCTTG TAGAGGTAGT GGAACCCCGACCGGAGGGTC GGTCGGTAGC ATTGTGACAC 240 AAGGTTTCTT TAACAATATT ATCAACCAAGCTGGTAATGG TTGCGCGGGG AAAAGATTCT 300 ACACCCGTGA CTCTTTCGTT AACGCCGCTAATACTTTCCC CAACTTTGCC AATTCTGTTA 360 CCAGACGTGA AATTGCTACC ATGTTTGCTCATTTCACTCA CGAGACCGGA CATTTCTGCT 420 ACATAGAAGA GATTAACGGA GCAACACGTAACTACTGCCA GAGCAGCAAC ACACAATACC 480 CATGTGCACC GGGAAAAGGC TACTTCGGTCGTGGTCCGAT CCAACTATCA TGGAACTACA 540 ACTACGGAGC GTGTGGTCAA AGTCTCGGTCTTGACCTTCT ACGCCAGCCC GAACTTGTGG 600 GTAGCAACCC AACTGTAGCT TTCAGGTCGGGTTTGTGGTT TTGGATGAAT AGCGTAAGGC 660 CGGTTCTGAA CCAAGGGTTT GGAGCCACCATTAGAGCTAT TAATGGAATG GAATGTAACG 720 GTGGTAATTC CGGTGCAGTC AACGCAAGGATTGGATACTA TAGAGACTAT TGTGGACAGC 780 TTGGTGTGGA CCCTGGTCCT AACCTTAGTTGCTAAAAAAC CTTTGAACCC AAACACGGAC 840 ATATGTGACG TGGCATGTAA TAAGTGAGATATACTAAAAT TTCACACGTA TGTACTTTAT 900 GTCGGGTCTC GGTGTTCCCT GCGTCACAAGCAAAAAATTG TTGTAATAAA CTTGTGAAAG 960 TGATTTTCTT TTCTTATGTG ACTTCTTATGAAAGAGAATT TTAAGATTGA TAGATGTTTG 1020 AGTTTGACAC TTGCAGTATC TGATTTTGGGATACTCGCAT AAAAAAAAAA AAAAAAAAA 1079 (2) INFORMATION FOR SEQ ID NO: 38:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 952 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: AAAACAACATAACATGGCTT CCACCAAAAT TTCCTTAGTC TTTTTCCTTT GCCTCGTAGG 60 TCCTTGTATAGGCGCTGGAA CCACAAGCGC TGCCGAATCA GTCAGTAGCA TTGTGACACA 120 AGGTTTCTTTAACAACATTA TCAACCAAGC CGGTAATGGC TGCACGAGAA AAAGATTCAA 180 CACCCGCGACTCTTTCATTG ACGCTGCTAA TAATTTTCCA AACTTTGCAA GTTCCGTTAC 240 AAGACGTGAAATTGCAAGCA TGTTTGCTCA TGTCACTCAC GAGACCGGAC ACTTCTGCTA 300 CATAGAAGAAATAAAGAAAA GGTCACGTGG TAGGTGCGAC GAGAACGTAG AGCAGAGACC 360 ATGTCCATCACAGAGTAAAG GTCACTCCGG TCGTGGTCAT CAGAGTCTCG GTCTCGACCT 420 TCTACGCCAGCCTGAGCTTG TGGGTAGCAA CCCAACTGTA GCTTTCAGGA AGGGTTTGTC 480 GTTTTGGATTAATAGCGTAA GGCCGGTGCT GAACCAAGGA TTTGGAGCCA CTATAAGAGC 540 CATCAATGGGATGGAATGTA ACGGTGGGAA TTCAGGTGCG GTCAAGGCAA GGATTGGGTA 600 CTATAGAGACTATTGTAGAC AGCTTGGTGT GGATCCTGGT CATAACCTTA GTTGCTAAAA 660 CGTTTTACAAACGCATACAA CGTGACGTGT CACGTGATAA TGCGGAATAA GAAGTTACAT 720 TTCAAACAGGGCAAGGATTC AGAATCGCAA GTACATGAAT TAACATTCTG TAACTGTTAA 780 TTGTATCAAGTTGTGTTAAT ATATAAGAGC TAGGAGAATT GGTCTTTCTT ATGTCTAGCT 840 AGTAACCATCTATGTTTGTA GCTAGGAGCT ACTTGTTGTT TCTAGTTGTT TGTCTATCTA 900 AGCCAATCATACAAAATCTA TGCTTATTCA CAAAAAAAAA AAAAAAAAAA AA 952 (2) INFORMATION FORSEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1250 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GAATTCGGCA CGAGCACACA CAATCCTGCC TATAGCCACC ATGTCTCCTC AGCCTACCTT 60AGCCACCACC ACCACGCCTG CTACTGGGCC TAACCCTTCC TATTACCAAA CAGCTCCTGT 120TAGCCAAGAG AGTATCCAGC GAACGGAGGT TCTTCTCCAC CTGTACGCCT ACCAGCACAC 180CCAAGGAAAA CCAAATGCTA ACCAGACTGT CATAGTGGAT CCAAAGCTCC CCGCGTGTTT 240CGGTGCCCTT GCTGCTAATG ACTGGACCAT ATATGATGGC ACTTGCACCA ATGCAAACCT 300TGTTGCGCAC GCTCAAGGTT TGCACATTGG TGCTGGTATG ACCAAAGAAA ATTGGTTCAT 360TTGTTTCAAC ATGGTGTTTG TGGACCGAAG GTTTATGGGT TCCAGCTTCA AGGTGATGGG 420AGATTTTCGA GGAAACGAAG GTGAATGGGC AATTGTTGGT GGGACGGGAG AGTTTGCATA 480TGCACAAGGT GTCATAACCT TTAACAAGAC CTGGTCGGCC CAGGCAAATG TCAGGGAGCT 540TCATGTTCGT GCTTTGTGTC TGTCCTTCTC AAAAGCACCG GAAACACCGT GCTCAAGGAC 600ACCACGGGAG AGCTCTGTCA CCAAGATCGG CCCATGGGGT AAAATAAGTG GAGAATTTCT 660TGACGTTCCC ACGACACCAC AACGTCTAGA GTGTGTGACC ATTCGCCATG GAGTTGTCAT 720TGATTCACTT GCATTTTCCT TCGTCGACCA AGCTGGTGGA CAACATAACG TTGGCCCATG 780GGGTGGGCCA TGCGGGGACA ACAAGGACAC GATTAAACTT GGTCCATCGG AGATTGTGAC 840AGAAGTCTCT GGAACGATTG GTGTATTTGG AGCAGCCAAT GTCGAGTACA ATGCCATAAC 900ATCACTAACC ATTACCACAA ATGTCCGCAC GTACGGGCCC TTTGGAGAAC CGCAGTGTAC 960TCGTTTCAGT GTTCCCGTGC AGGACAAAAG CAGCATCGTG GGTTTCTTCG TGTGCGCTAG 1020GAAATACGTG GAGGCGCTCG GGGTTTACGT GTGTCCACCT ATTTCAAACT AGTCCCGAAG 1080TGCTTCACAC TCGGTGTGCC TTCCACCTTT CTATGTTGTG CCAATAAAGT AGGTTATATA 1140CTTTTGGTGT AATGCTATGC CCCCCCCCAA GTGTGATGGT TTTCATATCA GCAAGAAGTT 1200TCTCTTGCTA CTATATGCAA AATAAAAAAA AAAAAAAAAA AAAACTCGAG 1250 (2)INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:340 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 40: AGCTCGNGTG GCGAACGGAC GAGGAGTTCG CCCGGGAGATGCTCGCCGGN CTCAACCCAC 60 ACATCATCAC CCGCCTGAAC GTGTTCCCTC CGAGGAGCACGNTTGAAGGG TACGGTGACC 120 AGACGAGCAA GATCACGGTG GAGCACATCC AACACAACCTCGGNAAGCTC AACGTCGACA 180 AGGCAATCGA CGCCAAGAGG CTCTTCATCC TAGACCACCACGACAACTTC ATGCCTCACC 240 TGCTAAAGAT CAACAGTCTC CCAAACACAT TCCGTCTTACGNCACCAGGA CGCTGCTCTT 300 CCTGCAAGAT GACGGGACTC TCAAGNCAAT CGNCATCGAG340 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 436 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 41: ACATCGAGAA GGGATCATGA ATTGACCCGA TCCCCAACTCAAGAACCGGA ATGGGCCACC 60 AAAATCCCCT AATGCTACTC TACCCCAACA CATCTGACATCGATGGTGAG AGCGCCACCG 120 GGATCACAGC CAAGGGCATC CCCAACAGCA TCTCCATCTGATCGATGTTA TGCCGTTTTA 180 TGTTATTGTT GTATGTTTCC CATTGCAAAT AAGGAACGCTGCATGTGCAC GTTTCATGAG 240 TGGCCAGAAG CGTGCTCGTT CACGTTGAGG CGAGTTGTGTTTATTTTCGT GTTGATGAAG 300 TGTTTTGGCC GCAATGGAAA GCCCGTGGGA TGGCACGGATGAAGGGTTGT GGTCGGCCAG 360 TGTGTTGAGC TGGACATATA TACGGAAAAA AATTGTATGAGACCGGGTCT CATAGACCCA 420 AAAAAAAAAA AAAAAA 436 (2) INFORMATION FOR SEQID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1162 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:AAGACACAAC CTTATTTTGC ATATAGTAGC AAGAGAAACT TCTTGCTGAT ATGAAAACCA 60TCACACTTGG GGGGGGGGCA TAGCATTACA CCAAAAGTAT ATAACCTACT TTATTGGCAC 120AACATAGAAA GGTGGAAGGC ACACCGAGTG TGAAGCACTT CGGGACTAGT TTGAAATAGG 180TGGACACACG TAAACCCCGA GCGCCTCCAC GTATTCCTAG CGCACACGAA GAAACCCACG 240ATGCTGCTTT TGTCCTGCAC GGGAACACTG AAACGAGTAC ACTGCGGTTC TCCAAAGGGC 300CCGTACGTGC GGACATTTGT GGTAATGGTT AGTGATGTTA TGGCATTGTA CTCGACATTG 360GCTGCTCCAA ATACACCAAT CGTTCCAGAG ACTTCTGTCA CAATCTCCGA TGGACCAAGT 420TTAATCGTGT CCTTGTTGTC CCCGCATGGC CCACCCCATG GGTCAACAGC CCACAGCTTC 480ACAAGCCAAT ACACACACCA ATATAGTCTC CTTTCCAGAG CTAGCTAGCA TCAATGGCTT 540CTGGTGCTCA GATGAAGATG GTTGCCGTCG GCATGATGCT GGCCATCATG TTCATCGCTG 600CAGCTCATGC AGAACCAGCG CCTGCAGAAA CTTGCATCGA CAAGACCGAA AAAGTTGGTC 660TTGCCACTGA CTGCATCTGC TCCAAGAACT GTGCTTGTGC AGGAAAGTGC ATCTTAGAAG 720GCGGCGAGGG TGGCGAAATC CAGAAGTGCT TTGTCGAATG CGTGCTGAAA AATGACTGCA 780ACTGCAATGC TAAGCACCAC AGTGCCGCAG CCCCTCAGTA AGGAGACTCC TGATTGGAGC 840TTCTTCAGCC ATGCTATGAG GTCTGCGTCC ACAAGTCCAC AGTCAACATT TGAGTAAATT 900AATAAACCTC TTATACCTGC GTTCTTGGCA TGCGCTTATT GCTTGCAGCG AATGTATGTT 960GAGAGTTGGT TTCCCGGTCA CAGCTCATAT ATATGCTGCC AGTCACGTGT ACGACGACTG 1020CCACATGTAT TGCTGGAAAT GAATATGTAT GTGTATTTGT CCTTTTCGTC ATTTGGGTCA 1080TTTTAAAAAT AAGTTGTATG CTGGAGCATG ACAACAAGTG TATTTGGCTA CTATAATATT 1140ATTAATGATA TGATGTCTTG TT 1162 (2) INFORMATION FOR SEQ ID NO: 43: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1371 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATCCACCTCGATCCAGCCAT GCTGTCCTCC AAGTCTTCTT TAGCTGTTGC TCTAATCCTT 60 GTGGTCACCCTGACAGATGT GCTACCGCTG GTATCTTCTT CACGTGGCCT CGTTGGGGTA 120 CCCACTGATGGGGCACTGGA GGACTCTCTG TTGATGATGG AGAGATTCCA TGGCTGGATG 180 GCAAAGCATGGCAAGTCGTA TGCGGGAGTC GAGGAGAAAC TGCGGCGGTT TGACATATTC 240 CGCAGGAACGTAGAGTTCAT CGAGGCGGCG AACCGAGATG GCAGGCTCTC GTACACCCTC 300 GGGGTGAACCAGTTCGCCGA CCTCACCCAC GAGGAGTTCC TTGCCACGCA CACCAGCCGC 360 CGTGTGGTGCCGTCAGAGGA GATGGTGATT ACAACTCGCG CTGGCGTTGT TGTCGAGGGT 420 GCCAATTGTCAGCCGGCGCC AAATGCTGTG CCTCGTAGCA TCAATTGGGT GAATCAAAGC 480 AAAGTCACCCCAGTCAAAAA TCAAGGAAAA GTATGCGGGG CTTGCTGGGC TTTTTCTGCC 540 GTGGCCACGATCGAAAGCGC CTACGCGGTC GCCAAGCGAG GCGAGCCGCC GGTTCTGTCC 600 GAGCAGGAGCTCATCGACTG TGACACAATC GACAGAGGCT GCACGAGCGG CGAGATGTAC 660 AATGCCTACTTCTGGGTCTT GAGGAACGGC GGCATCGCCA ACAGCTCAAC GTACCCCTAC 720 AAAGAGACTGACGGCAAGTG CGAGAGAGGG AAACTGCAGG AACACGCGGC CACGATCAGG 780 GACTACAAATTCGTCAAACC CAACTGCGAG GAGAAGCTCA TGGCAGCCGT GGCGGTGCGA 840 CCCGTCGCCGTCGGGTTCGA CTCCAACGAC GAATGCTTCA AGTTCTACCA AGCTGGTTTG 900 TACGACGGCATGTGCATCAT GCACGGGGAA TACTTTGGCC CGTGCTCGTC CAACGACCGC 960 ATCCACTCCTTGGCCATTGT CGGGTACGCC GGCAAGGGGG GCGACAGGGT CAAGTACTGG 1020 ATCGCCAAGAACTCGTGGGG CGAGAAGTGG GGAAAGAAGG GCTACGTCTG GCTGAAGAAG 1080 GATGTTGATGAGCCGGAAGG CCTCTGCGGC CTTGCAATTC AGCCGGTATA TCCTATAGTC 1140 TGATCTGATCTGACGAGATC GACTGCACTG GGCGTGCATG AAACCTACGG AAATGGCATT 1200 CACCTATATTTTGGGTTGCT CTGTATGCAT GGATGCGCCT ACTATATTTT ACTACATATA 1260 TATTCATCTCCCGCTAATAA AACTACATGT CCTTGTATCC ATTTATGCAC GTTTATCCAT 1320 ATCTTGAATAAATTGGATGG ATTGGTTATC CAAAAAAAAA AAAAAAAAAA A 1371 (2) INFORMATION FORSEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 723 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CAACCGGGAA CCGGAGGCCT CCGACCCCTC ACCTCGCTGT GGTTGACAAC ATCGACCTGT 60GCCCAAGACC TCGGGGAACG CTACCAACTC CGAACCCAAG TATGTCCCCG CCTTCGACGA 120GGCCGACGTC AAAGAAGGAG GTGAAGGGCG TTCTGTACCC ACGAGGCGAC GCACGTGTGT 180CAGTGGAACG GGCAGGCCAG GTCAAACGGC GGGCTCATCG AGGGGATCGC CGACTACGTG 240CGGCTCAAGG CCGACCTCGC GCCGACGCAC TGGCGCCCGC AGGGGAGCGG CGACCGTTGG 300GACGAGGGGT ACGACGTGAC GGCCAAGTTC CTGGACTACT GCGACTCCCT CAAGGCCGGG 360TTCGTGTCGG AGATGAACAG CAAGCTCAAG GACGGATACA GCGACGACTA CTTCGTGCAG 420ATCCTGGGGA AGAGCGTGGA CCAGCTGTGG AACGACTACA AGGCCAAGTA CCCCCCAGCC 480CCAGGGCTGA TCGACGATGC ATGCAGTTTG TTGTTGTATG TGTACCGGTC TTCGTCTACA 540TACAGATACA TTATAGTACT TGTATTACTG TACAATTTAT GTACTGCCTG GAATGGAATA 600AATCAGCGTT GGCACGGTGT GTGTTAACGA ATTGACGAGA CAAAGGGACC GTCTATAGGT 660CATGTCATCG GTTGCCTGAA ATACATTGAA CATCATCACT TTCTTTACAG CAAAAAAAAA 720AAA 723 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 168 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:Met Gly Phe Val Leu Phe Ser Gln Leu Pro Ser Phe Leu Leu Val Ser 1 5 1015 Thr Leu Leu Leu Phe Leu Val Ile Ser His Ser Cys Arg Ala Gln Asn 20 2530 Ser Gln Gln Asp Tyr Leu Asp Ala His Asn Thr Ala Arg Ala Asp Val 35 4045 Gly Val Glu Pro Leu Thr Trp Asp Asp Gln Val Ala Ala Tyr Ala Gln 50 5560 Asn Tyr Ala Ser Gln Leu Ala Ala Asp Cys Asn Leu Val His Ser His 65 7075 80 Gly Gln Tyr Gly Glu Asn Leu Ala Glu Gly Ser Gly Asp Phe Met Thr 8590 95 Ala Ala Lys Ala Val Glu Met Trp Val Asp Glu Lys Gln Tyr Tyr Asp100 105 110 His Asp Ser Asn Thr Cys Ala Gln Gly Gln Val Cys Gly His TyrThr 115 120 125 Gln Val Val Trp Arg Asn Ser Val Arg Val Gly Cys Ala ArgVal Gln 130 135 140 Cys Asn Asn Gly Gly Tyr Val Val Ser Cys Asn Tyr AspPro Pro Gly 145 150 155 160 Asn Tyr Arg Gly Glu Ser Pro Tyr 165 (2)INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Met Gly Phe LeuThr Thr Ile Val Ala Cys Phe Ile Thr Phe Ala Ile 1 5 10 15 Leu Ile HisSer Ser Lys Ala Gln Asn Ser Pro Gln Asp Tyr Leu Asn 20 25 30 Pro His AsnAla Ala Arg Arg Gln Val Gly Val Gly Pro Met Thr Trp 35 40 45 Asp Asn ArgLeu Ala Ala Tyr Ala Gln Asn Tyr Ala Asn Gln Arg Ile 50 55 60 Gly Asp CysGly Met Ile His Ser His Gly Pro Tyr Gly Glu Asn Leu 65 70 75 80 Ala AlaAla Phe Pro Gln Leu Asn Ala Ala Gly Ala Val Lys Met Trp 85 90 95 Val AspGlu Lys Arg Phe Tyr Asp Tyr Asn Ser Asn Ser Cys Val Gly 100 105 110 GlyVal Cys Gly His Tyr Thr Gln Val Val Trp Arg Asn Ser Val Arg 115 120 125Leu Gly Cys Ala Arg Val Arg Ser Asn Asn Gly Trp Phe Phe Ile Thr 130 135140 Cys Asn Tyr Asp Pro Pro Gly Asn Phe Ile Gly Gln Arg Pro Phe Gly 145150 155 160 Asp Leu Glu Glu Gln Pro Phe Asp Ser Lys Leu Glu Leu Pro ThrAsp 165 170 175 Val (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 47: GGGATCCCTG CA 12 (2) INFORMATIONFOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:ATAGTCTTGT TGAGAGTT 18 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 49: TCACGGGTTG GGGTTTCTAC 20 (2)INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 50: AGGAGATGGT TTGGTGGA 18 (2) INFORMATION FOR SEQ ID NO: 51: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: ATACGTTCTA CTATCATAGT 20(2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 52: Ala Thr Phe Asp Ile Val Asn Lys Cys Thr Tyr Thr Val TrpAla Ala 1 5 10 15 Ala Ser Pro Gly Gly Gly Arg Arg 20 (2) INFORMATION FORSEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Ala ThrPhe Asp Ile Val Asn Gln Cys Thr Tyr Thr Val Trp Ala Ala 1 5 10 15 AlaSer Pro Gly Gly Gly Arg Gln Leu Asn 20 25 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GGGATCCCTG CA 12 (2)INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 55: AATCCCATGG CTATAGGA 18 (2) INFORMATION FOR SEQ ID NO: 56: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: CCGGTACCGG CATG 14 (2)INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 57: GTTTATGTGA GGTAGCCATG GTGGGAAGAC TTGTTGGG 38 (2) INFORMATIONFOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:GATCGCGGTA CCGAGCTCCT CTAGGGGGCC AAGG 34 (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: GTTTATCTGAGGTAGCCATG GTGAGAAGAC TTGTTGGA 38 (2) INFORMATION FOR SEQ ID NO: 60: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: GATCGCGGTA CCGAGCTCCCTTGGGGGGCA AG 32 (2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 61: AATTTCCCGG GCCCTCTAGA CTGCAGTGGATCCGAGCT 38 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 62: CCGATCCACT GCAGTCTAGA GGGCCCGGGA 30(2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 63: CAAAACTCTC AACAAGACTA TTTGGATGCC C 31 (2) INFORMATION FORSEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:GAACTTCCTA AAAGCTTCCC CTTTTATGCC 30 (2) INFORMATION FOR SEQ ID NO: 65:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: CAGCAGCTAT GAATGCAT18 (2) INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 66: GGCAGGGATA TTCTGGC 17 (2) INFORMATION FOR SEQ ID NO: 67:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: TGCAAAGCTT GCATGCC17 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 68: ATGTTYGAYG ARAAYAA 17 (2) INFORMATION FOR SEQ ID NO: 69:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: AACATCTTGG TCTGATGG18 (2) INFORMATION FOR SEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 70: GGYTTRTCNG CRTCCCA 17 (2) INFORMATION FOR SEQ ID NO: 71:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: SCWTTCTRNC CCCAGTA17 (2) INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 72: GGRTKAKTMT AAAATTGNAC CCA 23 (2) INFORMATION FOR SEQ IDNO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: ACRAARCARTCRTGRAARTG 20 (2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 74: ATGTTYGAYG ARAAYAA 17 (2)INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 75: Met Phe Asp Glu Asn Asn 1 5 (2) INFORMATION FOR SEQ IDNO: 76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: AAYAAYTGYCCNACNAC 17 (2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Asn Asn Cys Pro Thr Thr 1 5(2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 78: ACRTCRTARA ARTCYTT 17 (2) INFORMATION FOR SEQ ID NO: 79:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Lys Asp Phe Tyr Asp Val1 5 (2) INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 80: CKRCARCART AYTGRTC 17 (2) INFORMATION FOR SEQ ID NO: 81:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: Asp Gln Tyr Cys CysArg 1 5 (2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 82: GCCTCGAGGA TCCTATTGAA AAAG 24 (2)INFORMATION FOR SEQ ID NO: 83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 83: GCCTCGAGTG CTTAAAGAGC TTTC 24 (2) INFORMATION FOR SEQ ID NO:84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: CTATGAATGCATCATAAGTG 20 (2) INFORMATION FOR SEQ ID NO: 85: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 85: GCGGAATTCA AAAAAAAAAA AAAACATAAG 30(2) INFORMATION FOR SEQ ID NO: 86: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 86: CCATAACAAA CTCCTGCTTG GGCACGGCAA GAGTGGGATA 40 (2)INFORMATION FOR SEQ ID NO: 87: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 87: TATCCCACTC TTGCCGTGCC CAAGCAGGAG TTTGTTATGG 40 (2)INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 88: GATCGAATTC ATTCAAGATA CAACATTTCT 30 (2) INFORMATION FOR SEQID NO: 89: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: CATTCTCAAGGTCCGG 16 (2) INFORMATION FOR SEQ ID NO: 90: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 90: CATCTGAATT CTCCCAACAA GTCTTCCC 28(2) INFORMATION FOR SEQ ID NO: 91: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 91: AACACCTATC GATTGAGCCC CTGCTATGTC AATGCTGGTG GC 42 (2)INFORMATION FOR SEQ ID NO: 92: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:81 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 92: GGATCCGTTT GCATTTCACC AGTTTACTAC TACATTAAAA TGAGGCTTTG 50TAAATTCACA GC TCT (2) INFORMATION FOR SEQ ID NO:93: (i) SEQUENCECHARACTERISTICS: (B) TYPE: nucleic acid (C) STRANDEDNESS: single (ii)MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: GTGACCGAGCTCAAAGAAAA ATACAGTACA ATA 33 (2) INFORMATION FOR SEQ ID NO: 94: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: ACCGTGGGAT CCACAGTAAAAAACTGAAAC TCC 33 (2) INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 95: GTGACCGGAT CCAAAGAAAA ATACAGTACAATA 33 (2) INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 96: ACCGTGGAGC TCACAGTAAA AAACTGAAAC TCC 33 (2)INFORMATION FOR SEQ ID NO: 97: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 97: GAACTCCAGG ACGAGGC 17 (2) INFORMATION FOR SEQ ID NO: 98: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 359 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 98: Met Ala Ala Ile Thr Leu Leu Gly Leu Leu LeuVal Ala Ser Ser Ile 1 5 10 15 Asp Ile Ala Gly Ala Gln Ser Ile Gly ValCys Tyr Gly Met Leu Gly 20 25 30 Asn Asn Leu Pro Asn His Trp Glu Val IleGln Leu Tyr Lys Ser Arg 35 40 45 Asn Ile Gly Arg Leu Arg Leu Tyr Asp ProAsn His Gly Ala Leu Gln 50 55 60 Ala Leu Lys Gly Ser Asn Ile Glu Val MetLeu Gly Leu Pro Asn Ser 65 70 75 80 Asp Val Lys His Ile Ala Ser Gly MetGlu His Ala Arg Trp Trp Val 85 90 95 Gln Lys Asn Val Lys Asp Phe Trp ProAsp Val Lys Ile Lys Tyr Ile 100 105 110 Ala Val Gly Asn Glu Ile Ser ProVal Thr Gly Thr Ser Tyr Leu Thr 115 120 125 Ser Phe Leu Thr Pro Ala MetVal Asn Ile Tyr Lys Ala Ile Gly Glu 130 135 140 Ala Gly Leu Gly Asn AsnIle Lys Val Ser Thr Ser Val Asp Met Thr 145 150 155 160 Leu Ile Gly AsnSer Tyr Pro Pro Ser Gln Gly Ser Phe Arg Asn Asp 165 170 175 Ala Arg TrpPhe Val Asp Pro Ile Val Gly Phe Leu Arg Asp Thr Arg 180 185 190 Ala ProLeu Leu Val Asn Ile Tyr Pro Tyr Phe Ser Tyr Ser Gly Asn 195 200 205 ProGly Gln Ile Ser Leu Pro Tyr Ser Leu Phe Thr Ala Pro Asn Val 210 215 220Val Val Gln Asp Gly Ser Arg Gln Tyr Arg Asn Leu Phe Asp Ala Met 225 230235 240 Leu Asp Ser Val Tyr Ala Ala Leu Glu Arg Ser Gly Gly Ala Ser Val245 250 255 Gly Ile Val Val Ser Glu Ser Gly Trp Pro Ser Ala Gly Ala PheGly 260 265 270 Ala Thr Tyr Asp Asn Ala Ala Thr Tyr Leu Arg Asn Leu IleGln His 275 280 285 Ala Lys Glu Gly Ser Pro Arg Lys Pro Gly Pro Ile GluThr Tyr Ile 290 295 300 Phe Ala Met Phe Asp Glu Asn Asn Lys Asn Pro GluLeu Glu Lys His 305 310 315 320 Phe Gly Leu Phe Ser Pro Asn Lys Gln ProLys Tyr Asn Ile Asn Phe 325 330 335 Gly Val Ser Gly Gly Val Trp Asp SerSer Val Glu Thr Asn Ala Thr 340 345 350 Ala Ser Leu Val Ser Glu Met 355(2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 765 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 99: AAATGACAAT TGAAATTTAT TAAAGTAATA TGATCTAATATGTTCAAACA AGCTCAATCA 60 CAATTTAATT TAATTAATAT TGACCGCGCA TCGCGCGAGTACGACTACTA GTTCAATTTA 120 GAAAGCTGGA GCCATTGCCG GAGTAATCAT GAAATTGGCGGAGTTGACTT CCGATTCCGG 180 TGATGGTGCG AGTATTCCCG AGCTTTTGAT CTCCGCCATTCCACGGCGTG CATTTCCACC 240 TTGTTCTTCA CACAATCTGG GAAGGTATAC ACCTTCTCCAGCAGCAAGGT TGAAGTAGAG 300 ATCAATAATA TTAGGGCAAT GTTTGTAGCA CTGAGGGGAGCACAGCTTCT GGGTAAAGTG 360 GCACTCGAGG AGAGAATCAG AAGAATTCCG AAAGTATTCCTATCAACACC GCAAGCCTGA 420 ATACACTGGT CGGTTTCAAT CCAGTCTTTG AGCTTATCAGCCTCAATTTC CGACGTCTTA 480 CATGTATATA CTTCTTCACC ACTCCTTTGG AGACGTTTCTCTAACACGCA ACGCTTCCCA 540 GTACTTGAAA TCGCAAAAGC GCATGAGTCC TTGTTTAGATTCTCGCATGT TATGCTCCCT 600 AGAGTGACTT GAACACAGAA GACAAGTGCA CAAGCAACAATAGCCAAAGT AAAGTTGTGG 660 AATGAAGCAA CCATGGTGAA AAATCTAGCA ACAATTGATCAGACTATAAA TTTTTTCTGA 720 GTATATATAG CTCCTTTTGG CTTTCGACTC TCCTTTTTTGTGGTT 765 (2) INFORMATION FOR SEQ ID NO: 100: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1567 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: GGCACGAGCATTTTCCACAT CTTCTGCCAC TTCTAATTCC AAACTTCCAG TTCGAGAAAT 60 CCCAGGAGACTATGGTTTCC CCTTTTGTGG AGCCATAAAA GATAGATATG ACTACTTCTA 120 CAACCTCGGCACAGACGAAT TCTTTCTTAC CAAAATGCAA AAATACAACT CTACTGTCTT 180 TAGAACCAACATGCCACCAG GTCCATTCAT TGCTAAAAAT CCCAAAGTAA TTGTTCTCCT 240 CGATGCCAAAACATTTCCCG TTCTTTTCGA CAACTCTAAA GTCGAAAAAA TGAACGTTCT 300 TGATGGCACGTACGTGCCAT CTACTGATTT CTATGGCGGA TATCGCCCGT GTGCTTATCT 360 TGATCCTTCTGAGTCAACTC ATGCCACACT TAAAGGGTTC TTTTTATCTT TAATCTCCCA 420 GCTTCATAATCAATTKATTC CTTTATTTAG AACCTCAATT TCTGGTCTTT TCGCAAATCT 480 TGAGAATGAGATTTCCCAAA ATGGCAAAGC GAACTTCAAC AATATCAGCG ACATTATGTC 540 ATTCGATTTTGTTTTTCGTT TGTTATGTGA CAAGACCAGT CCCCATGACA CAAATCTTGG 600 CTCTAATGGACCAAAACTCT TTGATATATG GCTGTTGCCT CAACTTGCTC CATTGTTTAG 660 TCTAGGTCKTAAAATTTGTG CCGAACTTTC TGGAAGATTT AATGTTGCAT ACTTTTCCCT 720 TGCCATTTTTTCTAGTGAGA TCGAATTACC AGAAGCTTTA TGATGCTTTT AGCAAGCATG 780 ACTTAGTTTTTCTTGCAGGT TTCAATGCTT ATGGTGGGAT GAAAGTTTTA TTCCCTGCAC 900 TGATAAAGTGGGTCGCCAAT GGAGGAAAGA GTTTACACAC TCGGCTGGCA AATGAAATCA 960 GGACANTTATCAAAGAAGAA TGTGGGACCA TAACTCTATC AGCAATCAAC AAGATGAGTT 1020 TAGTAAAATCAGTAGTGTAT GAAGTATTAA GAATTGAACC TCCAGTTCCA TTCCAATATG 1080 GTAAAGCCAAAGAAGATATC ATAATCCAAA GCCATGATTC AACTTTCTTA GTCAAGAAAG 1140 GTGAAATGATCTTTGGATAT CAGCCTTTTG CTACAAAAGA TCCAAAGATT TTTGACAAAC 1200 CAGAGGAGTTTATTCCGGAG AGGTTCATGG CCGAAGGGGA AAAATTATTA AAGTATGTGT 1260 ATTGGTCAAATGCAAGAGAG ACAGATGATC CAACGGTGGA CAACAAACAA TGCCCAGCGA 1320 AAAATCTTGTCGTGCTTTTG TGCAGGTTGA TGTTGGTGGA GGTTTTCATG CGTTACGACA 1380 CATTCACAGTGGAGTCAACY AAGCTCTTTC TTGGGTCATC AGTAACGTTC ACGACTCTGG 1440 AAAAAGCGACATGAGTTTCA GATATCTTAA TTGTAGGCTG CKAATAATAA TGTGGTCATT 1500 CTGCKAATTATTGTACTTGT GCTGATGTAC TTGACTTCGA GTGGATATAA TAATGCACTG 1560 TTTTTAG 1567(2) INFORMATION FOR SEQ ID NO: 101: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 392 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 101: GGTCACTAAT ACCAAACCAA GATTCAGTTG GTTGAATGATGATTTAACAG TTTAAGACAT 60 TAGACTATAC ATGTGATTAA GTACTAGTAC TTCCCCCACTCGCGAGGAAG TACATTCAAC 120 CAGAAGGAGA CCTCCAGAGT TAGTTCAGCA GNTCATTTATCTACATAAAC TACAACTCTA 180 AGCAAGACAT AAGGGATAAA CAGGAGTATA CATATTATACTACACATATT ACTGCGCAAT 240 AGCAAGTGGA CACAAAGGTA CCACTATTTG GTAGTGTTAGGGGCGTTCCC AAAAGGGGTT 300 CGGTCCCTTG AGTCATTGGA GGCTGTCTTG ATCTCTTGGCAGTTGGACAA TTCCACAATT 360 ACCTGAACTT CGCCAATTGG TTGAGATGTG AA 392 (2)INFORMATION FOR SEQ ID NO: 102: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 396 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 102: TGCCGCTTTA AGTGTGCTCG AACCTCCTAC TGGTAATGAGGATGATGATG ACCTGGAATT 60 TGAAAATGTC CACTGGAATG GTTCAGATAT GGCATCCGATGATACTCAAA AATCTCATAG 120 ACCAAGGCAC CGCGTACATA AATCGTCTGG TTCCCACAAGGNCATGAGCC GCTCCCTTTC 180 ATGTGACTCG CAATCAAAAG GATCTATTTC TACACCTCGTGGGTCCATGG TTGACCTAAG 240 CAAACTCGAG ATGGCTGCAC TGTGGAGATA TTGGCGACACTTTAACCTTA GGGAGGTATT 300 CCTAACCCTC GAAAGAGCAC TTATTGATGT GGTCAGAGCATTCANATCTC AGAAATGGGC 360 GAGTGGAGGT ATTGTGGATT GGTCAAGCTG CAAGAG 396(2) INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 653 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 103: CTTGTTTGGT TGTTTGAGTT ATTTTGCTTC TAAGAACTTTGTGAGAAATG GCTGCTAACG 60 ATGCTACTTC ATCCGAGGAG GGACAAGTGT TCGGCTGCCACAAGGTTGAG GAATGGAACG 120 AGTACTTCAA GAAAGGCGTT GAGACTAAGA AACTGGTGGTGGTCGATTTT ACTGCTTCAT 180 GGTGCGGSCC TTGCCGTTTT ATTGCCCCAA TTCTTGCTGACATTGCTAAG AAGATGCCCC 240 ATGTTATATT CCTCAAGGTT GATGTTGATG AACTGAAGACTGTTTCAGCG GGAATGGAGT 300 GTGGAGGCAA TGCCAACTTT TGTCTTCATT AAAGATGGAAAAGAAGTGGA CAGAGTTGTT 360 GGTGCCAAGA AAGAGGAGTT GCAGCAGACC ATAGTGAAGCATGCTGCTCC TGCTACTGTC 420 ACTGCTTGAA TCTCCTTAAT CAAGGGGATG ATATCCCATATTTAGTAGTA TTGTCTTTTG 480 TAATAACCAA GTAACTTGTT CGAATTTCAC ACTATGGATCACTGTATGGT TGTACTATCC 540 ACCATGTTTT TATTGCTTTT GTGAACCTTG TCTTGTTGCTTGGAATCTGA TTTGTGCATT 600 ACTGGTGTAA GGCTATATGC CCAATTCYAC AAAAAGACTACTTTTAGATT TCT 653 (2) INFORMATION FOR SEQ ID NO: 104: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1697 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: ATTCAAAATTATTGGATCCT AGTTTTAAGT GGAACTCCTA ATTAATCAAT CATTTCATGA 60 TCAACCTTTACTAACATCAC CATTTTCCTT GACAACATTA TTTTCCGACT TACCATTTTC 120 CTTAACACCATTACCCTCTG GATTTTCCTT GACAATATTA CTCTCCGACT TCTCATTTTC 180 GTTCACTTTATCAGTAATCT TGGCTTCTTC AGCAGCTGCT TCTTTCTCTT TCTCTTTCTC 240 TTTCTCAGCCCTTTCTCTTT CTTCTTCCTC AGCCTTCAAT TTTGGCTCCT CCTTTGCAGT 300 TTCAAGAGCTTTAATCAGTC TCTCCAAACA AGTGTTTGCA TTTTCCTTAG AAGACTTGGG 360 CATTAAATTCTCAGCAACAT CAGCAGGAGT AATATTAGTT TCCCCCAATA AATGACGAAT 420 CTCAGGAAAATGATCATGAG ACTCAATATC TAGATAATTA TTTGCAAGCA CCTTGAAGGA 480 GTCAAAGCAACAGTATGATA ACACAATGTG TTTATCCATC CTCCCCCTCC GAATTAAAGC 540 AGGGTCAAGCTTTTCCACAA AGTTGGTAGT GAAAACAATA AGCCTTTCAC CACCAATAGC 600 TGACCATAACCCATCAATAA AGTTCAAAAG CCCAGATAAA GTCACCTCGC TTTGCTTTTT 660 CTCCTCTCGATTTTTCATCT TCTCCTTGAC GGCATCTTTC TCGTCTTTTA CTTCCTCTTC 720 CTTGTCGTCTTTCTTCTTCT CCCTTTGGCC GGTAAGGTCA AGCGAACAGT CGATGTCTTC 780 AATCACAATGATAGACTTAC TAGTAGTATC TATTAATAAC TTTCTTAGCT CGGTGTTGTC 840 CTTAACCGCTGTCAATTCAA GATCATAGAC ATCATATTGT AAGAAGTTAG CCATTGCAGC 900 AATCATGCTAGACTTACCGG TTCCTGGAGG ACCATATAGA AGATAACCAC GCTTCCATGC 960 CTTGCCAATCTTGGCATAAT AGTCTTTTGA CTTGCTAAAT GTTTGAAGGT CATCCATAAT 1020 CTCTTGTTTCTTGTTTGGCT CCATGGCTAA AGTATCAAAT GTTGATGGAT GTTCAAACAC 1080 TACTTGGCTCCACATTCTCC TCCTGTATCC ATACCCACCA TCTCCCTTAC TGTTTGTGTA 1140 CAACTTTCTCTGCCTTTCTC TTACTGAAAT TGCCTTCCCT TCGTCCAATA CATACTTCAA 1200 GTATGAAGCGGTGATAAGCT CGCGGTTCTT TCTGTGAAAC TTGAGTTTGA AATACCTCTT 1260 CTCATCCTCCCTAGGGTACC AAGAAATTGT CTGTCTGCTG GCTACTTGTT GGCTAGAAAT 1320 CCACCAGACTTTCTCGCCTT TATATTCATC GGTTACCTCC TCATGATCAT CCATGGTTAG 1380 TACAAGAGATTGGCCATCTT TCACTACATT GGCTTTGAGA CGCTTAGCTT GTGTGGAGGA 1440 GTTCTTGCTTAGGTACCTTT CAATTGCTAC ATAAGCTTTG CTACGCTCGA ACCAGCCATC 1500 AGTTTCATACTCATGAAAAA TAATGTGCAT ATAAGGGTAG AAATAGCTCA CGAGTTTATC 1560 GGTATACCTCCTAATATGAC CACGAAGTTC GTGAGGAAAA TAGTTCTGGT ACATGGTCCA 1620 GGCAAACATGATTGTTGCAA TAGCTGGACC CAACTGAGTC CAAACATCTT GCATCATCAT 1680 CATCATCTAATTTCTCT 1697 (2) INFORMATION FOR SEQ ID NO: 105 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 654 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: TTAACCTTGAGATAAGCATT AAAAAAACTC AATGGCAGGG AAGGTTGAGA AAGTGCTTGC 60 AGTACTGATGCTTGCAATGC TTCTGTTTTC GGAGCATTTC ATGGCTGCTA ATCATGAAAT 120 TAAAACAACTGAAGATAACT CTATTAGCCC TTTCTGCTTA ATAAAATGTT TATATGGATG 180 CAGGGGGTTGCCACCTGCAA AAGCAGCCAT TTGTGCAGCT CAATGTTTGT TTAAGTGCGC 240 TGTCCAAGATGAGGCCAATA TAGCTGAAAC TAAGGGCATA ATAGGTGAGA CTGCATACAA 300 CCAGTATGATGTTGGATGTG CCCTTGGCTA CTGCTCTGAG TTCCTGTTGA ATTATGATGA 360 GAGGAGGTTCAACTGCTGCA TGGAATACTG TCGCGAGGGC AAAATGACCT GTCCTGTTGA 420 GGCTGCACCTTGAAGAAATG GTTGCCCTAA AATTATCGCC TCATCAAATG GAAGTACACT 480 GCTTTTTCTACTTCCGGTGT TTAGTAGTAG TAGTAAATAA GTGAGGCATG TTACGTACTC 540 TTATGTTTTGTAATAATTAT GCTTTTTAAT AATGTAATCT GTCTGTGTGC ATACAATGCA 600 CACGACGCTAGCTACTACTT TTTATCTACT AAAAACGAAA AGTAACTTAT TTCT 654 (2) INFORMATION FORSEQ ID NO: 106: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1031 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQID NO: 106: GAGACAAATA CCTATAATAA GCCATTCATA ATCTTCTTGC TTCTTGTTCAGAATAATGGG 60 GAATCTTTTC TGTTGCGTGC TTGTGAAGCA ATCAGATGTT GCGGTCAAGGAGAGATTTGG 120 CAAATTCCAA AAAGTACTTA ATCCAGGTCT CCAATTTGTT CCATGGGTCATCGGTGATTA 180 CGTCGCCGGT ACACTGACCC TTCGTCTTCA GCAACTCGAT GTTCAGTGTGAAACCAAAAC 240 AAAGGACAAT GTGTTTGTGA CAGTGGTTGC ATCCATACAA TACAGAGTCTTAGCTGACAA 300 GGCAAGTGAT GCTTTTTACA GACTCAGCAA TCCAACCACC CAAATCAAAGCCTACGTCTT 360 TGATGTGATC AGAGCATGTG TTCCAAAGCT GAACTTGGAC GATGTGTTCGAGCAGAAGAA 420 TGAAATTGCC AAATCTGTGG AAGAAGAGCT AGACAAAGCC ATGACTGCTTATGGTTACGA 480 AATCCTTCAA ACCCTAATTA TCGACATTGA GCCTGATCAA CAGGTTAAACGTGCCATGAA 540 CGAAATCAAC GCCGCGGCGA GGATGAGAGT GGCAGCGAGC GAAAAAGCAGAGGCTGAGAA 600 AATCATTCAG ATCAAAAGAG CAGAGGGTGA AGCAGAGTCA AAGTACCTGTCGGGACTCGG 660 AATCGCTCGG CAGAGACAAG CGATCGTGGA CGGTCTTGAG AGACAGTGTTCTTGGGTTCG 720 CAGGAAACGT GCCAGGGACG TCAGCGAAGG ATGTGTTGGA CATGGTGATGATGACTCAGT 780 ACTTTGACAC AATGAGAGAT ATCGGAGCAA CTTCTAAATC CTCTGCGGTGTTTATCCCTC 840 ACGGTCCAGG CGCCGTCTCT GACGTGGCAG CGCAGATTCG AAATGGATTATTACAGGCCA 900 ACAATGCCTC CTAATCACTC AAGTCAAATT GTCTTGGTCG TCTCTTTATATATTTTCGTA 960 TCTTCTTATT AAAAAGGTAA ATTTGACTTT TAATATAATG GTGTGCTTATTGCGAAAAAA 1020 AAAAAAAAAA A 1031 412 (2) INFORMATION FOR SEQ ID NO:107: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 107: Met Phe Ser Lys Thr Asn Leu PheLeu Cys Leu Ser Leu Ala Ile Leu 1 5 10 15 Val Ile Val Ile Ser Ser GlnVal Asp Ala Arg Glu Met Ser Lys Ala 20 25 30 Pro Ala Ser Ile Thr Gln AlaMet Asn Ser Asn Ile Ile Thr Asp Gln 35 40 45 Lys Met Gly Ala Gly Ile ThrArg Lys Ile Pro Gly Trp Ile Arg Lys 50 55 60 Gly Ala Lys Pro Gly Gly LysIle Ile Gly Lys Ala Cys Lys Ile Cys 65 70 75 80 Ser Cys Lys Tyr Gln IleCys Ser Lys Cys Pro Lys Cys His Asp 85 90 95 (2) INFORMATION FOR SEQ IDNO: 108: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 108: Met Phe Ser Lys Thr Ile Leu PheLeu Cys Phe Ser Leu Ala Ile Leu 1 5 10 15 Val Met Val Ile Ser Ser GlnAla Asp Ala Arg Glu Met Ser Lys Ala 20 25 30 Ala Ala Pro Ile Thr Gln AlaMet Asn Ser Asn Ile Ile Thr Asp Gln 35 40 45 Lys Thr Gly Ala Gly Ile IleArg Lys Ile Pro Gly Trp Ile Arg Lys 50 55 60 Gly Ala Lys Gly Gly Asn IleIle Gly Lys Ala Cys Lys Ile Cys Ser 65 70 75 80 Cys Lys Tyr Gln Ile CysSer Lys Cys Pro Lys Cys His Asp 85 90 (2) INFORMATION FOR SEQ ID NO:109: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 109: Met Phe Ser Lys Thr Asn Leu PheLeu Cys Leu Ser Leu Ala Ile Leu 1 5 10 15 Leu Ile Val Ile Ser Ser GlnAla Asp Ala Arg Gln Ile Ser Lys Ala 20 25 30 Ala Ala Pro Ile Thr His AlaMet Asn Ser Asn Asn Ile Thr Asn Gln 35 40 45 Lys Thr Gly Ala Gly Ile IleArg Lys Ile Pro Gly Trp Ile Arg Lys 50 55 60 Gly Ala Lys Pro Gly Gly LysVal Ala Gly Lys Ala Cys Lys Ile Cys 65 70 75 80 Ser Cys Lys Tyr Gln IleCys Ser Lys Cys Pro Lys Cys His Asp 85 90 95 (2) INFORMATION FOR SEQ IDNO: 110: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 110: Met Phe Ser Lys Thr Asn Leu PheLeu Cys Leu Ser Leu Ala Ile Leu 1 5 10 15 Leu Ile Val Ile Ser Ser GlnAla Asp Ala Arg Glu Met Ser Lys Ala 20 25 30 Ala Val Pro Ile Thr Gln AlaMet Asn Ser Asn Asn Ile Thr Asn Gln 35 40 45 Lys Thr Gly Ala Gly Ile IleArg Lys Ile Pro Gly Trp Ile Arg Lys 50 55 60 Gly Ala Lys Pro Gly Gly LysVal Ala Gly Lys Ala Cys Lys Ile Cys 65 70 75 80 Ser Cys Lys Tyr Gln IleCys Ser Lys Cys Pro Lys Cys His Asp 85 90 95 (2) INFORMATION FOR SEQ IDNO: 111: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 111 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 111: Met Phe Ser Lys Thr Asn Leu PheLeu Cys Leu Ser Leu Ala Ile Leu 1 5 10 15 Leu Ile Val Ile Ser Ser GlnAla Asp Ala Arg Glu Thr Ser Lys Ala 20 25 30 Thr Ala Pro Ile Thr Gln GluMet Asn Ser Asn Asn Thr Thr Asp Gln 35 40 45 Lys Ile Pro Lys Arg Pro LysPro Gly Gly Asn Ile Phe Gly Lys Ala 50 55 60 Cys Lys Ile Cys Pro Cys LysTyr Gln Ile Cys Ser Lys Cys Pro Lys 65 70 75 80 Cys Asp Asp Gln Asn IleAla Gly Lys Phe Cys Lys Ile Cys Ser Cys 85 90 95 Lys Thr Gln Ile Cys SerLys Cys Pro Lys Cys His Asn Gln Asn 100 105 110

What is claimed is:
 1. An isolated DNA molecule comprising a nucleotidesequence that encodes a polypeptide having β-1,3-glucanase activity,wherein said polypeptide comprises the amino acid sequence encoded bySEQ ID NO:
 20. 2. A chimeric gene comprising a nucleic acid promotersequence operatively linked to the isolated DNA molecule of claim
 1. 3.A vector comprising the chimeric gene of claim
 2. 4. A transgenic plantcell comprising the chimeric gene of claim
 2. 5. A transgenic plantcomprising the transgenic plant cell of claim
 4. 6. Seed from thetransgenic plant of claim
 5. 7. The isolated DNA molecule of claim 1,wherein nucleotide sequence comprises SEQ ID NO:20.